用毛细管电泳分离蛋白质

SDS-capillary gel electrophoresis (SDS-CGE), also called capillary sieving electrophoresis

(CSE) or capillary gel electrophoresis (CGE), shows many advantages over classical SDS-

PAGE. These advantages include on-column detection, automated operation, great resolving

power, and capability of accurate protein quantification and molecular weight determination

[3–8]. The first papers on CGE were published in the 1980s [9, 10]. As in slab-gels, agarose

and cross-linked polyacrylamide (CPA) was used as sieving matrices, and these matrices

were prepared directly inside the capillary columns. In the early 1990s [11], linear

polyacrylamide (LPA) was introduced to replace CPA, but an in-capillary polymerization

procedure was still used for the gel preparation. The lifetimes of these columns were limited

(usually less than 10 runs) [12], and the run-to-run reproducibility was poor. Currently,

replaceable and water-soluble linear or slightly branched polymers, such as linear

polyacrylamide [11–13], poly(ethylene glycol) [11], poly(ethylene oxide) [14], dextran [15–

17], pullulan [18, 19] and cross-linked polyacrylamide [20–22] are used as sieving matrices

for CGE [5, 11, 23–25]. Availability of these polymer matrices has led to improved

reproducibility and robustness of this methodology.

Recently, CGE has been recognized and established [26] as an important tool in

biopharmaceutical industry to support analytical characterization, process development, and

quality control of therapeutic recombinant monoclonal antibodies (rMAbs) [26–29]. In an

effort to make CGE-based methods accepted by biotechnology companies, scientists in

various pharmaceutical industries and regulatory authorities conducted cross-laboratory

research to examine the reliability and robustness of the method [30, 31]. It is expected that

some CGE methods will soon be used in pharmaceutical and biotechnological industries. In

light of this advancement, we write this paper to review briefly the progress of CGE for

protein analysis. We focus mainly on the methodology and application aspects of CGE. In

the methodology aspect, we review the common sieving matrices, wall coatings, and

detection strategies used in CGE. CGE performed in microfabricated channels and CGE as

one dimension in two-dimensional (2D) separations are also discussed. In the application

aspect, we present a few separations related to or closely related to practical uses. Table II

provides a summary of literatures on CGE of proteins based on the sieving matrices used.2. Methodology

The basic apparatus for CGE is identical to that of capillary zone electrophoresis (CZE) and

consists of a capillary column, an on-column detector, and a high voltage power supply. The

major difference between the two techniques is the separation media: a sieving matrix is

employed in CGE while a background electrolyte solution is utilized in CZE.

2.1. Sieving Matrices for CGE

Polyacrylamide (PA ) has been widely used in slab gel electrophoresis of proteins, and

consequently it is frequently utilized in CGE. Initially, PA gels were synthesized in-situ

inside capillaries [10, 32, 33]. Typically, a capillary column was prepared by mixing

acrylamide (monomer), N,N’-methylenebis(acrylamide) (Bis, cross-linker), ammonium

peroxy-disulfate or ammonium persulfate (radical initiator), N,N,N’,N’-

tetramethylethylenediamine (TEMED, catalyst) and other background electrolytes,

introducing the mixture into the capillary, and allowing the solution to polymerize inside the

capillary. While this worked in general, problems occasionally arose when PA shrank during

polymerization, breaking PA gel into segments and/or forming bubbles inside the column.

Additionally, a good column could work well for only the first a few runs, as large

molecules and particulate materials accumulated at the injection end of the column, which

deteriorated and eventually shut down the separation.

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To address this issue, a replaceable sieving polymer – a low viscous LPA solution – was prepared. This sieving matrix was successfully used for DNA sequencing [34], as well as for protein sizing [12]. Because the sieving polymer inside a separation column could be replaced after each run, the run-to-run reproducibility was improved considerably.It should be noted that, when LPA was developed, its low viscosity (or replaceability) was emphasized. This might be why CPA was rarely investigated as a replaceable sieving matrix initially, because common sense tells that a cross-linked polymer would have a high viscosity. In 2005, Lu et al. [35] noticed that, if the degree of cross-linking was carefully controlled, CPA was an excellent replaceable sieving matrix – superior over LPA for protein separations in many aspects. Using this sieving matrix, CGE was capable of resolving proteins ranging from ～4–250 kD in less than 20 min.When PA sieving matrices are used to run CGE, capillary walls often need to be coated for achieving high quality separations. Poly(N,N-dimethylacrylamide)-grafted PA , a derivative of PA, was prepared by Zhang et al. [36] in 2006, and when this polymer was used to sieve proteins, capillary wall coating could be avoided. This is because poly(N,N-dimethylacrylamide)-grafted PA is capable of coating capillary walls dynamically.Various polysaccharides form another important type of sieving matrices for protein separations. One advantage of polysaccharides is that these polymers do not absorb as much UV light as PA does. Ganzler et al. [11] separated SDS-protein complexes using dextran and poly(ethylene glycol) (PEG). The separated proteins were detected at 214 nm in which dextran and PEG are transparent. These matrices had moderate viscosities and could be conveniently replenished. Luo et al. [17] performed high-throughput protein analysis by multiplexed SDS-CGE, and Xu et al. [37] realized separation and characterization of SDS-protein complexes on a microchip with UV adsorption detection using similar matrices.Hydroxypropyl cellulose (HPC) is another polysaccharide sieving matrix used in CGE. For example, Hu et al. [38] developed a CGE-laser-induced fluorescence (LIF) method for

separating proteins from HT29 cancer cells. Pullulan [24, 39, 40] and hydroxyethyl cellulose

[41] were used for CGE, as well.

Other polymers have also been utilized for protein sieving. Yu et al. [42] used poly(vinyl

alcohol) (PVA) to perform on-line protein concentration and separation. Bernard et al. [43]

used poly(2-ethyl-2-oxazoline) for CGE and achieved separation efficiencies of ～10 million

plates per meter. Hu et al. [8] used polyethylene oxide (PEO) to analyze the protein contents

in a single HT29 cancer cell and obtained protein profiles similar to those determined by

other methods.

Using dynamic light-scattering, Sumitomo et al. [44] evaluated the mesh size and

homogeneity of three sieving polymer solutions, PA , PEO and HPC. Based on their

experimental results, these authors concluded that an optimal sieving polymer for separating

proteins ranging from 14.3 to 97.2 kD is a homogeneous polymer network with a mesh size

of less than 10 nm. Sumitomo et al. also stated that PEO in solution can aggregate, degrade

into smaller pieces, and form polymer–micelle complexes with SDS. This disturbs protein–

SDS complexation and impairs the protein separation efficiency. Recently, the same group

surveyed the composition of the separation buffers, and results showed that Tris-CHES

buffer was able to suppress SDS adsorption to PEO and achieve separation of six proteins

[45].

Commercial sieving kits are now available to run CGE. These kits are largely from

Beckman-Coulter (https://www.wendangku.net/doc/879014970.html,), Agilent Technologies (https://www.wendangku.net/doc/879014970.html,)

and Bio-Rad Laboratories (https://www.wendangku.net/doc/879014970.html, ) and they are optimized for their CGE

instruments. Beckman SDS Gel was demonstrated to be capable of sizing membrane

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proteins [46], protein biotoxins [47], and antibodies [48], but coated capillaries or channels

were usually needed to achieve good separations [49]. Using Bio-Rad CE-SDS run buffer,

Na et al. [50, 51] used uncoated fused-silica capillaries for CGE and separated poly(ethylene

glycol)-modified proteins. This kit was also employed for quantitative analysis of antibodies

[52], RuBisCo in spinach [53], and water-/salt-proteins from bovine and ostrich meat [54].

Agilent commercialized the first microchip capillary electrophoresis system (Agilent 2100

Bioanalyzer), along with its microchips. Agilent 2100 Kit was provided with this instrument

and utilized for analysis of half-antibody [55] and glycoproteins by microchip CGE [56, 57].

In these applications, the gel was pipetted into the designated reservoirs on a chip and

propelled, by use of a syringe, into the chip channels.

2.2. Capillary Coatings

The interior walls of capillaries used in CGE are often coated for two purposes: reducing

protein-wall interactions and suppressing electroosmotic flow (EOF). An uncoated wall can

interact with proteins electrostatically (if part of the protein molecule is positively charged)

and/or hydrophobically (if a portion of the protein molecule is hydrophobic), and these

interactions deteriorate separation efficiencies. In CGE, the strengths of these interactions

are greatly reduced because proteins have reacted with SDS forming hydrophilic and

negatively charged SDS-protein complexes. Therefore, wall coating in CGE is used

primarily for EOF suppressions.

Running CGE at low or zero EOF is important for achieving reproducible results. If one

uses an uncoated capillary to run CGE, the EOF will carry the sieving matrix from anode to

cathode while SDS-protein complexes migrate in the opposite direction. Some of the

proteins will never pass the detector, unless the EOF is so large that it brings all SDS-protein

complexes to the detector. Usually, this condition cannot be guaranteed. Another problem

associated with EOF is its instability as the wall conditions change. The fluctuation of EOF

causes the migration time change, and subsequently the separations become irreproducible.

Including an internal standard in samples can mitigate this problem, as long as the standard

does not interfere with protein peak detections. Pugsley et al. [58] developed a dye

(fluorescently-labeled aspartic acid) that worked well as an internal standard, because it

migrates faster than all fluorescently-labeled SDS-protein complexes.

Numerous approaches have been explored to control/suppress EOF, and the most commonly

used approach is to derivatize capillary walls via either dynamic coatings [59–62] or

covalent coatings [63–65]. Progress in the field of polymeric coatings can be found in a

number of reviews [66–68].

A dynamic coating, due to its simplicity, is a convenient way to modify capillary wall

properties. It is normally produced by putting appropriate additives (often polymers) into

SDS-SGE run buffers (or sieving matrices) and flushing the capillary columns with these

run buffers before separation. Several polymers, including polydimethylacrylamide [61],

epoxy poly(dimethylacrylamide) [69–71], and poly(-hydroxyethylacrylamide) [62] were

used to create a dynamic coating. The exposure of silica surfaces to very dilute solutions of

these polymers causes development of dense polymer layers via hydrogen bonding,

electrostatic attractions and/or hydrophobic forces [72]. The molecular weight of the

polymer has a strong impact on the stability of the coating since the adhesive forces/energies

per chain increase in proportion to the number of monomer units [73]. Some CGE sieving

matrices are excellent dynamic coating additives [8, 51, 58, 74]. With these matrices, bare

capillaries can be used directly for protein separations.

Covalent coatings are generally more stable than dynamic coatings. These coatings are

obtained by chemically bonding desired substances to capillary walls. One of the most

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common coating protocols was introduced by Hjerten in 1985 [65]. Typically, 3-

(trimethoxysilyl)propyl methacrylate is first attached to a capillary wall, leaving acrylic

groups exposed on the wall surface. The capillary is then filled with a polymerizing solution

containing acrylamide and a polymerization initiator. The free acrylic groups attached to a

capillary wall serve as anchors for growing linear polymer chains. A problem of this coating

is that linear molecules cannot cover the capillary wall completely. The poorly covered area

will adsorb proteins and create EOF. To improve this situation, a CPA coating was

developed by Gao and Liu in 2004 [75] and successfully used for SDS-CGE [35].

2.3. Microfabricated channels for CGE Microfabricated (or microchip) devices are developed with a goal to perform and integrate multiple analytical processes (e.g. sample pretreatment, solution distribution/mixing,separation, detection, etc.) on a chip platform [76, 77]. Due to the short column length and high separation efficiency, microchip CGE is generally fast, typically from a few seconds to a few minutes. Yao et al. [78] is recognized as the first who performed SDS-PAGE in a microfabricated channel, and the separations were completed in less than 1 min. By combining an on-chip dye staining with an electrophoretic dilution step (similar to a destaining step), Bousse et al. [79] obtained excellent resolutions for microchip CGE of proteins. On the basis of this work, the first commercial microchip instrument was constructed by Agilent Technologies. In 2004, Han et al. [22] and Herr et al. [80] applied an in-channel photopolymerization approach to prepare CPA gels inside a microchip channel for SDS-PAGE, and a separation speed of <30 s per run was demonstrated. These authors also prepared a gradient CPA gel for on-chip protein sizing [20] and successfully implemented sample pre-concentration using these photo-patterned gels [21]. Huang et al.[81] combined isotachophoresis (ITP) to concentrate proteins for subsequent CGE. Xu et al.[82] performed on-line electrokinetic supercharging preconcentration on a microchip to improve method sensitivity. Tsai et al. [83] tested simultaneous separations of both native and SDS-denatured proteins on a single microchip with 36 microchannels. Herr et al. [84]

recently integrated saliva pretreatment (mixing, incubation, and enrichment) with

subsequent quantitative immunoassays and measured the concentration of endogenous

MMP-8 in saliva. More recently, He and Herr [85] photopatterned different gels inside a

microfluidic chamber for protein immunoblotting. Fig. 1 presents the immunoblotting chip.

Gel-separation was first performed in the vertical dimension, and the separated proteins

were then transferred to the immunoblotting gel in the horizontal dimension. Electric fields

were applied to the chamber via the parallel microchannels, and the microchannel arrays

were designed such that uniform electric fields were produced over the chamber area during

separation and transfer steps in both the vertical and horizontal dimensions.

In 2005, Fruetel et al. [47] reported a hand-held microchip instrument called μChemLab?

that is capable of performing CZE and CGE in parallel. The instrument consisted of a

microfluidics module, a dual channel LIF detection module, an integrated multichannel

high-voltage power supply, and a main control board containing the laser diode drivers, user

interface, and an embedded microprocessor (see Fig. 2A). It has an approximate volume of

7×8×4.5 cubic inches (see Fig. 2B).

Microchip devices were originally fabricated on glass substrates [86, 87]. Over the past

decade, polymeric chips have attracted growing attention, due to the low material and

fabrication costs. Polystyrene [88], polyesters [88], polycarbonate [89],

poly(dimethylsiloxane) (PDMS) [90] and poly(methyl methacrylate) (PMMA) [91] were

used to fabricate microchips. Hybrid materials are also used [49]. All these chips have been

tested for CGE separation of proteins. Performance of microchip-based gel electrophoresis

has been compared with that of capillary-based gel electrophoresis [41, 56, 57, 92–94]. In

general, the performances are comparable, while microchip CGE provides faster separations.

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2.4. CGE as One Separation Dimension in Two-Dimensional Protein Analyses

2D separation techniques are powerful tools for protein analysis, because the peak capacity of a 2D analysis is the multiplication product of the peak capacities of the two individual dimensions. To realize this resolving power, Chen et al. [90] constructed a 2D separation device using reconfigurable PDMS slabs in 2002. Four slabs were used to make channels and reservoirs to perform the first dimensional (1st -D) separation – isoelectric focusing (IEF). Then, the middle two slabs containing the IEF-resolved proteins were inserted into another two pieces of slabs which contained multiplexed channels for the second dimensional (2nd -D) separation – CGE. Because of their elastomeric nature, PDMS slabs could be attached and detached reversibly without fluid leaking. Although 2D separations were performed, high resolving power was not demonstrated using this device.Griebel et al. [19] fabricated 300 parallel channels (64 mm long × 50 μm wide × 50 μm deep) on a PMMA chip. A 50-μm opening was produced at one end of chip across all these parallel channels. To prepare for the separation, these parallel channels were filled with 15% (w/v) pullulan. IEF (the 1st -D separation) was performed first on a separate device – a conventional immobilized pH gradients (IPG) strip. After IEF, the IPG strip was brought to the opening on the chip for parallel CGE (the 2nd -D) separations. However, 2D separation results were not disclosed in this paper.IEF and CGE were incorporated in the above devices, but they were coupled in an off-line fashion. To implement on-line integration, Li et al. [89] integrated IEF with CGE on a polycarbonate microchip using PEO as their sieving matrix. Fig. 3 presents the channel layout of this chip: one horizontal channel intersected by eight parallel vertical channels.The IEF was performed in the horizontal channel, and SDS-PEO gel electrophoresis was performed in the vertical channels. Preferably, an SDS-PEO sieving matrix should be filled in the vertical channels before IEF was performed. However, the device as designed had a limitation in this regard. Because the 1st -D and the 2nd -D channels were directly connected,

the SDS in the SDS-PEO matrix in the 2nd -D channels would bleed into the 1st -D channel

due to molecular diffusion and electric field distortion at the channel intersections during

IEF. The presence of SDS in the 1st -D channel would bind to proteins (which would add

negative charges on the proteins), and therefore ruin the IEF. To circumvent this problem,

the authors filled the 2nd

-D channels with a matrix containing PEO but not SDS. The SDS required for the 2nd -D separation was electrokinetically introduced to the matrix after IEF

was complete. During the SDS introduction, the protein bands focused based on their p I

values were diffused/broadened before they were conjugated with SDS and

electrokinetically injected to the 2nd -D channels. Thus, some IEF resolution was lost.

In 2008, Liu et al. [95] carried out IEF and parallel SDS gel electrophoresis on a similar

device. PA gel plugs were patterned via photopolymerization at various locations to stop

hydrodynamic flows between reservoirs/channels and thus prevent unwanted bleeding/

mixing. These gel plugs may cause problems when channels require frequent washing.

It should be noted that the concept of this 2D separation chip had been discussed earlier

[96], but 2D separation results were never published.

In a separate effort, Yang et al. [97] combined capillary isoelectric focusing (CIEF) with

CGE in a linear format (see Fig. 4A) via a polyethersulfone dialysis hollow fiber interface.

Fig. 4B illustrated the detailed structure of this interface. After hemoglobin variants were

focused in the CIEF capillary, the catholyte in the reservoir on the methacrylate plate was

replaced by a CGE buffer. The CGE buffer also served as a chemical mobilization solution

for the CIEF. As voltages were applied to both capillaries, CIEF-resolved protein bands

were chemically mobilized to the hollow fiber. At the same time, negatively charged SDS

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continuously migrated into the hollow fiber and reacted with the proteins (forming SDS-

protein complexes), and the SDS-protein complexes were subsequently injected into the

CGE capillary for the 2nd -D separation. Because the CIEF-resolved proteins were

continuously injected into the CGE capillary, some of the resolving power was sacrificed.

Additionally, Michels et al. [18] coupled CGE (the 1st -D) with MEKC (the 2nd -D) by

connecting the exit-end of a CGE capillary to the sampling-end of an MEKC capillary. A

small gap was left between the two capillaries and filled with an MEKC running buffer to

facilitate electric field application and sample injection for MEKC. Two high voltage (HV)

power supplies were used in this work. HV1 was used to execute sample injection and

separation, and HV2 was utilized for MEKC. After a period of initial CGE separation, a

fixed length (e.g., 10 s migration) of CGE-resolved protein band(s) was allowed to enter the

gap. HV2 was turned on to apply a potential to the gap solution so that there was no electric

field across the CGE capillary (to stop the CGE), while an electric field was created across

the MEKC capillary to inject the proteins in the gap into the MEKC capillary and execute

the MEKC separation. [Note: HV1 was on all the time.] When the MEKC separation was

complete, HV2 was turned off for a given period of time (e.g ., 10 s) so that more CGE-

resolved proteins entered the gap. Then, HV2 was turned on again to execute the sample

injection and MEKC separation. These operations were repeated until the proteins inside the

CGE capillary were exhausted. This separation technique was successfully applied for

separations of proteins from bacterium Deinococcus radiodurans [18] and proteins from

single mammalian cells [40]. The method was later modified, and the separation speed was

improved from 3–5 h per run to ～1 h per run [98].

In 2006, Shadpour et al. [91] incorporated CGE with MEKC on a PMMA device. Fig. 5A

shows the channel layout of the microchip. By applying a vacuum to reservoir D while

reservoirs E and F were sealed, a sieving matrix was aspirated into d 1 channel from

reservoir C . As soon as the sieving matrix reached point d 2 (as shown in Fig. 5B), the

vacuum on reservoir D was removed. An MEKC buffer was pressurized into d 3 channel

from reservoirs F while reservoirs A -C were sealed. A protein mixture was injected into d 1for CGE. As the first protein peak approached point d 2, appropriate voltages were applied to

various reservoirs to stop CGE and effect MEKC. After MEKC was complete, voltages on

the reservoirs were changed to stop MEKC and resume CGE for a short period of time (e.g.,

0.5 second) to allow a fraction of CGE-resolved protein band to migrate toward point d 2.

These operations were repeated until all CGE-resolved proteins were separated by MEKC.

Complex proteins samples were analyzed using a similar chip and approach [99].

2.5. Detection Strategies

UV absorption is the most commonly used detection mode in CE, including CGE [15, 100,

101]. Proteins can be detected easily by a UV absorbance detector, because the peptide

bonds between amino acids and aromatic side groups in protein molecules absorb UV light

at 200–220 nm and 280 nm, respectively. Owing to the limited optical path length, the

concentration sensitivities of UV absorption detection are normally low, especially when

narrow capillaries are used.

LIF detectors are commonly used in CGE to improve concentration sensitivities. When an

LIF detector is used, proteins need to be fluorescently “labeled”. Proteins have been

covalently labeled by fluorescent dyes, such as naphthalene ?2,3-dicarboxaldehyde (NDA)

[102], 3-(2-furoyl) quinoline ?2- carboxaldehyde (FQ) [8, 103, 104] and

fluoresceinisothiocyanate (FITC) [105, 106]. In 2007, Michels et al. [107] reported an

improved fluorescent derivatization method for proteins analysis by CGE. In this assay,

rMAbs were derivatized with FQ in the presence of cyanide (CN ?). This technique NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

minimized sample preparation artifacts and greatly improved detection sensitivity of FQ-labeled rMAbs.Covalent labeling method has an intrinsic problem. A protein molecule usually has a number of sites that can react with a fluorescent labeling dye. Because these sites have different reactivities, it is challenging to make all sites to be labeled with the dye. This labeling reaction produces a mixture in which some proteins are un-labeled, some are fully-labeled,while the majority is partially-labeled. This mixture will cause peak-broadening or even multiple peaks in CE separations [108]. A postcolumn labeling method is often a good approach to address this problem. In 2009, Kaneta et al. [16] reported a postcolumn derivatization method for CGE separations of proteins. The method used a labeling dye of naphthalene-2, 3-dicarbaldehyde in the presence of 2-mercaptoethanol which played a role of a reducing agent in the derivatization reaction. Recently, these researchers replaced 2-mercaptoethanol with ethanethiol as the reducing agent and improved the method limits of detection by 1.4- to 4.5-fold [109].Alternatively, proteins can be dynamically labeled with fluorescent dyes [110]. In 2001, Jin et al. [111] showed that SDS-protein complexes could be dynamically labeled with NanoOrange. NanoOrange does not fluoresce much in aqueous solutions, but as it binds to a protein-SDS complex, it fluoresces substantially. Sano et al. [112] took a similar approach for CGE analysis of collagenase. Chiu et al. [74] labeled proteins with SYPRO Red and accomplished LIF detection using a low-cost He-Ne laser. In 2007, Wu et al. [113]developed an elegant approach for protein labeling. First a pseudo SDS dye was synthesized by attaching an alkyl chain to an ionic fluorescent dye (e.g., FITC). Since the long carbon chain is equivalent to the dodecyl group while the negatively charged fluorescent group resembles the sulfate group of SDS, the pseudo-SDS dye has the same function as SDS when binding to proteins. As a mixture of SDS and pseudo-SDS dye reacts with proteins,protein molecules are dynamically labeled with some pseudo-SDS dye. Fig. 6 presents a

schematic demonstration of pseudo-SDS dye-protein-SDS complex. Because each protein

can be associated with many pseudo-SDS dye molecules, the detection sensitivity can be

improved considerably. Using this approach, these authors obtained an LOD of 0.13 ng/mL

and a dynamic range of ～5 orders of magnitude for CGE analysis of BSA.

Fluorescence imagers have also been used as detectors for SDS-CGE [19, 90, 114, 115]. A

fluorescence imager is a great tool for early stage technology development since it allows

researchers to see the migration of proteins inside a capillary or a microfabricated channel.

The imaging area depends on the field of view of the imager but normally it will be about a

few millimeters to a few centimeters in diameter.

Mass spectrometers (MS) are excellent detectors, because they are capable of identifying

proteins. Coupling CGE with an MS, however, is challenging, because MS does not

normally have access to CGE resolved proteins. In addition, the SDS in the sieving matrix

interferes severely with MS detection. To address these issues, Lu et al. [116] developed an

approach to couple SDS-CGE with matrix-assisted laser desorption ionization time-of-flight

MS (MALDI-TOF-MS). Fig. 7 presents a schematic diagram of the experimental setup for

this work. Basically, a PTFE membrane was used to collect CGE-resolved proteins (so that a

MS detector will have access to these proteins). [Note: The collected proteins were actually

SDS-protein complexes that could not be analyzed directly by MS.] After the collection, the

SDS-protein complexes on the membrane were washed using an optimized solution to

remove SDS while proteins were retained on the membrane. After SDS removal, a MALDI

matrix was introduced onto the membrane for MALDI-TOF-MS analysis.

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3. Applications

In the literatures we surveyed, a lot of the papers still dealt with standard (or commercially-

purchased) proteins (see Table II). Here, we discuss only a few representative papers closely

related to practical applications.

3.1. Proteins in Biological Fluids

Analysis of proteins in biological fluids is challenging due to the complexity of sample

media. CGE offers a powerful tool to analyze these samples. In 2000, Lin et al . [46] used

CGE to analyze erythrocyte membrane proteins in blood samples. The erythrocyte

membrane samples were extracted from washed red cells, and spectrin in the samples was

removed before CGE run. Erythrocyte membrane proteins in normal red cell indices or from

healthy blood donors were utilized as controls. The same samples were analyzed by both

CGE and SDS-PAGE, and similar profiles were obtained.

In 2008, Obubuafo et al. [117] analyzed thrombin, an important marker for various

hemostasis-related diseases and conditions, by affinity microchip CGE for human plasma

samples and also for rabbit plasma sample. The method employed a PMMA microchip and

used LPA as sieving matrix. Two fluorescently labeled aptamer affinity probes, HD1 and

HD22, which bind respectively to thrombin exosites I and II were investigated. HD22

affinity assays of thrombin produced baseline-resolved peaks with favorable efficiency due

to its higher binding affinity, whereas HD1 assays showed poorer resolution of the free

aptamer and complex peaks. Therefore, HD22 was selected in determining the level of

thrombin in human plasma.

In 2011, Debaugnies et al. [118, 119] evaluated an automated CGE system, the Experion

instrument from BioRad, for its ability to separate and quantify the erythrocyte membrane

proteins. The major erythrocyte membrane proteins were extracted and purified from

membrane ghosts by centrifugation, immunoprecipitation and electroelution. Analyses were

performed using SDS-PAGE and SDS-CGE to establish a separation profile of the total

ghosts. As the SDS-CGE method was able to achieve the same conclusion as with SDS-

PAGE, except for the patient with elliptocytosis, Debaugnies et al. concluded that the new

SDS-CGE method could be proposed as an automated alternative method to the labor-

intensive SDS-PAGE analysis. Kaneta et al. [109] applied CGE with postcolumn

derivatization/LIF detection to analyses of two biological samples, namely a cell lysate and

a milk sample.

3.2. Proteins in Food Products

Monitoring food safety and food quality has become increasingly important in recent years.

Sample preparations are essential for these analyses. To examine the quality of seafood

products, Sotelo et al. [120] applied CGE for analysis of myofibrillar proteins in fish and

squid muscles. A Beckman-Coulter P/ACE 2000 capillary electrophoresis system was used

in this work, and the manufacturer recommended procedure was followed. Myosin and actin

contents in fish and squid muscles were measured, and these results were comparable to the

results from a slab-gel SDS-PAGE system. While the resolving powers of the two methods

were comparable, CGE had two significant advantages – automated operations and short

separation times. However, P/ACE 2000 could only analyze one sample per run. When a

batch of samples was to be analyzed, a technician could run all of them in a slab gel in one

run, and the differences between samples were readily recognized by direct lane-to-lane

comparisons. If these samples were analyzed serially by CGE, results comparisons were not

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Meat quality can be indicated by the profile and quantity of water-soluble and salt-soluble

proteins. Vallejo-Cordoba et al. [54] employed CGE and analyzed these proteins in bovine

and ostrich meats. Briefly, meats were mixed with water or saline buffer (typically, 0.6 M

NaCl/0.01 M phosphate buffer, pH 6.0, 0.5% polyphosphates), blended and centrifuged. The

filtered supernatant, sample buffer, benzoic acid (as internal standard) and mercaptoethanol

were mixed, boiled and then cooled down. Proteins in this sample were injected for CGE

analysis. CGE separations were carried out on a Bio-Rad CE system (BioFocus 3000), and

the manufacturer recommended protocols were followed. Profiles and concentrations of

water-soluble and salt-soluble proteins were measured successfully in this work.

Gomis et al. [121] analyzed cider proteins and determined their relative molecular masses.

Various methods were hired to isolate cider proteins for CGE [122]. Chiu et al. [74]

described a segmental filling method for analysis of SYPRO Red labeled SDS-proteins by

CE-LIF with electroosmotic counterflow of PEO. This method was capable of determining

casein in cow’s milk below 0.5 mM.

3.3. Proteins in Agricultural Products

RuBisCo accounts for more than 50% of the soluble protein in chloroplasts and is a key

enzyme in the photosynthetic fixation of carbon dioxide [123]. An accurate measurement of

the quantity of RuBisCo subunits would provide an indication of a plant’s physiological

status. Nicolas et al. [53] established a CGE method for analysis of RuBisCo in Spinach

leaves. To prepare samples for this method, spinach leaves were freshly harvested and

ground in a chilled mortar with a portion of inert sand and some chilled buffer (100 mM

Tris–hydrochloride, 0.1 mM EDTA and 1 mM ascorbic acid at pH 8.0). The homogenate

was centrifuged, and the supernatant was desalted. This sample was diluted 1:1 with the CE-

SDS protein sample buffer (CE-SDS Protein Kit: Bio-Rad, Hercules, CA, USA), and

benzoic acid was added as an internal standard (CE-SDS Protein Kit) to a final

concentration of 50 μg/mL. After SDS-protein complexes were formed, the sample was

ready for analysis. An HP3D capillary electrophoretic system (Hewlett-Packard,

Wilmington, DE, USA) was used in the work.

Chen et al. [124] also analyzed RuBisCo from leaves of Vigna unguiculata. Leaf tissues

were ground to a fine powder in liquid nitrogen. Proteins were extracted from leaf tissue at

0–4 °C in 80 mM Tris buffer containing 0.1 M P-mercaptoethanol, 2% (w/v) SDS, and 15%

(v/v) glycerol. The extract was centrifuged and the supernatant was used for protein

analysis. CGE was performed with a Bio-Rad 3000 system. Proteins in soybean seeds were

also analyzed using CGE by Gerber et al. [125]. Blazek and Caldwell [93] compared SDS-

CGE with the lab-on-a-chip technology to quantify the relative amount of 7S and 11S

fractions in twenty different soybean cultivars.

Marchetti-Deschmann et al. [126] recently evaluated a one-step single-grain wheat

extraction process followed by a CGE-on-a-chip analysis for fast and reliable wheat variety

control [119]. Based on the results of 15 different wheat varieties grown in Austria,

Marchetti-Deschmann et al. concluded that the CGE-on-a-chip system was a promising

alternative for the time-consuming and labor-intensive SDS-PAGE for high-throughput food

analysis.

3.4. Proteins in Clinical and Pharmaceutical Studies

Recombinant immunoglobulin G4 (IgG4), as well as other IgG antibodies, is made up of

two light chains and two heavy chains. In a normal human IgG4, disulfide bonds are formed

between a light chain (L) and a heavy chain (H), and also between two HL dimmers. In an

abnormal IgG4, there are no disulfide bonds between HL dimmers (the dimmers are linked NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

together by only noncovalent interactions). Vasilyeva et al. [55] used an Agilent 2100

Bioanalyzer to quantitate these HL dimmers of abnormal IgG4 in rMAb samples. The

microchip method described in this paper was suitable for analyzing samples containing HL

from approximately 0.6% to at least 5.2% (may be extended up to 80%). The LOD and limit

of quantitation (LOQ) were determined to be 0.05% and 0.59%, respectively. Good

correlations were obtained between this method and conventional SDS-PAGE, and between

this method and reversed-phase HPLC.

With the increasing therapeutic use of rMAbs, Analyzing the quality and purity of rMAbs

becomes an important and routine task for rMAb manufacturers. Hunt and Nashabeh [26]

developed a CGE method for analysis of rMAbs in biopharmaceutical industry. The method

included precolumn protein labeling, CGE separation and LIF detection. 5-

carboxytetramethylrhodamine succinimidyl ester was used as a labeling reagent. CGE

separations were performed on a Bio-Rad BioFocus 3000 CE system equipped with a LIF

detector. This method was validated according to the guidelines of the International

Committee on Harmonization and had been used as part of a control system for the release

of an rMAb pharmaceutical in Genentech, Inc. This method was optimized recently [30].

Guo et al . [52] developed a non-reduced SDS-CGE method and used it to study disulfide

heterogeneity in IgG2 antibodies. This method was proved to be a powerful tool to get

information on the backbone structure of IgG molecules. Zhang et al. [48] optimized a

similar method to analyze mAb1 drug substance under both reduced and non-reduced

conditions. Lancher et al. [127] established a generic method for monitoring disulfide

isomer heterogeneity in IgG2 antibodies, and applied this method for purity analysis of

reduced and non-reduced IgG2 mAbs [128]. Rustandi et al. [129] reported a wide range of

applications of CGE for mAb product development, including purity analyses for product

release, product-related impurities during process and cell-culture development, and product

stability evaluation. Cherkaoui et al. [130] used CGE to evaluate the IgG structural integrity

under various reduction conditions and track antibody reduction fragments.

Carbonyl-modified proteins are considered markers of oxidative damage in aged tissues and

diseases such as Parkinson’s, diabetes, emphysema, and atherosclerosis [131, 132]. Feng et

al. [133] developed a carbonyl detection method based on the reaction of Alexa 488

hydrazide with carbonyls and on the separation of the Alexa 488-labeled compounds by

CGE with a sheath flow cuvette. Because carbonyls on lipids, carbohydrates, and nucleic

acids could also react with Alexa 488 [134], yielding products that would interfere with the

detection of carbonyl-modified proteins, the Alexa 488-labeled proteins were further labeled

with another fluorogenic reagent – FQ. FQ only reacted with proteins, and its fluorescence

showed little spectral overlap with that of Alexa 488. Therefore, protein peaks with

fluorescence characteristics of both Alexa 488 and FQ belonged to carbonylated proteins.

The method was adequate for analyzing nanogram protein samples with femtomole levels of

carbonyls.

Mellado et al. [135] described an application of CGE for the analysis of rotavirus virus-like

particles. Particle’s apparent molecular masses and quantities were determined, and these

results were validated by comparing them with those obtained from traditional SDS-PAGE

and MALDI-TOF-MS.4. Conclusions

In conclusion, CGE is a powerful tool for protein analysis. Automated operation and short

separation times are two most significant advantages of CGE over conventional slab gel

electrophoresis. Reproducibility is still a shortcoming for CGE, although a lot of progress

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

has been made. Currently, CGE separations are performed usually in series, which makes

lane-to-lane comparisons not as convenient as in multilane slab gel electrophoresis [120,

136]. Microchip CGE is a promising platform for high speed protein analysis. At the time

being, however, most practical applications have been conducted using capillary-based

systems. While UV absorption detection is still a popular detection scheme for CGE, LIF

detection is gaining a lot of ground. The reason might be that reliable and affordable

fluorescence labeling dyes are commercially available, and that multiple labeling is less an

issue for CGE. CGE has been used as a separation dimension for 2D separations, but so far

the resolving power of these schemes could not compete with that of conventional 2D gels.

In terms of practical application, CGE has already been utilized for quality control and

purity test of monoclonal antibody products. Other imminent applications include clinical

diagnosis, food quality monitoring, etc. We expect CGE to be an important analytical

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Biographies

Mr. Zaifang Zhu earned his bachelor’s degree of Science in chemistry from Lanzhou

University (Lanzhou, P. R. China). He is currently a Ph.D student in the Department of

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Chemistry and Biochemistry at the University of Oklahoma. His research is on exploiting

capillary-based systems for bioanalysis.Ms. Joann J. Lu received her Master’s degree from Texas Tech University in 1994. Ms. Lu worked as a research associate and scientist at Bayor in West Heaven, Connecticut, Inhale Therapeutic in San Carlos, California, and Oculex Pharmaceuticals in Sunnyvale, California.She is now a Research Scientist in the Department of Chemistry and Biochemistry at

University of Oklahoma. Her research is focused on protein analysis.Professor Shaorong Liu received his Ph.D. degree from Texas Tech University in 1995.After worked as a postdoctoral fellow at Northeastern University in 1996 and University of California at Berkeley in 1997, he joined Molecular Dynamics in Sunnyvale, California as a Scientist in 1998 and Manager of Technology Development in 2000. Dr. Liu joined Texas Tech University as an Associate Professor in 2002, and Professor in 2007. Since 2008, Dr.Liu is a Professor in the Department of Chemistry and Biochemistry at University of Oklahoma. His research is focused capillary electrophoresis and microfluidic devices for high-speed and high-throughput bioanalysis.

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Fig. 1. Immunoblot chip

(a) Schematic design of the immunoblot chip for analysis of native proteins. The sample (2),

sample waste (3), buffer (1, 4, 5, 6) and buffer waste (7, 8) reservoirs are indicated in sketch

(not to scale). The middle region of the device (indicated as Chamber) has a length of 1.5

mm, a width of 1 mm and a depth of 20 μm. (b) Three separate zones inside the Chamber to

facilitate protein immunoblotting: a large-pore-size protein loading gel on the top, a smaller-

pore-size protein separation gel on the bottom-left and an antibody-functionalized blotting

gel on the bottom-right. Colored dyes were used to visualize the different gel regions.

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Fig. 2. μChemLab? instrument

(a) The μChemLab instrument with the top off showing the separation platform, the control

panel, the back of the LCD display, and the battery pack. The instrument is approximately

7″×8″×4.5″ and weighs 6 lbs. (b) The separation platform houses the microfluidic chip in a

compression manifold that connects the chip to eight fluid reservoirs, two sample injection

ports, and a LIF detection module. The overall size of the platform is approximately

5″×3″×4″. Reprinted from ref. [48] with permission.

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Fig. 3. Schematic of a plastic microchip for 2-D protein separation

Reprinted from ref. [90] with permission.

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蛋白质的盐析与透析

蛋白质的盐析与透析 一、实验目的 1.了解蛋白质的分离纯化方法 2.掌握蛋白质的盐析及透析方法 二、实验原理 在蛋白质溶液中加入一定浓度的中性盐，蛋白质即从溶液中沉淀析出，这种作用称为盐析。盐析法常用的盐类有硫酸铵、硫酸钠等。 蛋白质用盐析法沉淀分离后，需脱盐才能获得纯品，脱盐最常用的方法为透析法。蛋白质在溶液中因其胶体质点直径较大，不能透过半透膜，而无机盐及其它低分子物质可以透过，故利用透析法可以把经盐析法所得的蛋白质提纯，即把蛋白质溶液装入透析袋内，将袋口用线扎紧，然后把它放进蒸馏水或缓冲液中，蛋白质分子量大，不能透过透析袋而被保留在袋内，通过不断更换袋外蒸馏水或缓冲液，直至袋内盐分透析完为止。透析常需较长时间，宜在低温下进行。 三、实验材料和试剂 10%鸡蛋白溶液，含鸡蛋清的氯化钠蛋白溶液，饱和硫酸铵溶液，硫酸铵晶体，1%硝酸银溶液。 四、实验步骤 （一）蛋白质盐析 取10%鸡蛋白溶液5ml于试管中，加入等量饱和硫酸铵溶液，微微摇动试管，使溶液混合后静置数分钟，蛋白即析出，如无沉淀可再加少许饱和硫酸铵溶液，观察蛋白质的析出； 取少量沉淀混合物，加水稀释，观察沉淀是否会再溶解。 （二）蛋白质的透析 注入含鸡蛋清的氯化钠蛋白溶液5ml于透析袋中，将袋的开口端用线扎紧，然后悬挂在盛有蒸馏水的烧杯中，使其开口端位于水面之上。 经过10分钟后，自烧杯中取出1ml溶液于试管中，加1%硝酸银溶液一滴，如有白色氯化银沉淀生成，即证明蒸馏水中有Cl-存在。 再自烧杯中取出1ml溶液于另一试管中，加入1ml 10%的氢氧化钠溶液，然后滴加1-2滴1%的硫酸铜溶液，观察有无蓝紫色出现。 每隔20分钟更换蒸馏水一次，经过数小时，则可观察到透析袋内出现轻微混浊，此即为蛋白质沉淀。继续透析至蒸馏水中不再生成氯化银沉淀为止。 实验报告记录透析完毕所需的时间。 附：胶棉半透膜的制备 市售5%的胶棉液，加入干燥的150mL锥形瓶中，将锥形瓶横斜不断转动，使瓶的内壁和瓶口都均匀沾有胶棉液。倒出多余的胶棉液，然后倒置约1min使乙醚、乙醇不断蒸发，直到干燥。逐步剥离瓶口的薄膜，沿瓶壁薄膜夹缝注入蒸馏水，使薄膜逐步跟瓶壁胶离，轻轻取出，浸入蒸馏水中备用。 如有侵权请联系告知删除，感谢你们的配合！

毛细管电泳的基本原理及应用

毛细管电泳的基本原理及应用 摘要：毛细管电泳法是以弹性石英毛细管为分离通道，以高压直流电场为驱动力，依据样品中各组分之间淌度和分配行为上的差异而实现分离的电泳分离分析方法。该技术可分析的成分小至有机离子、大至生物大分子如蛋白质、核酸等。可用于分析多种体液样本如血清或血浆、尿、脑脊液及唾液等，比HPLC 分析高效、快速、微量。 关键词：毛细管电泳原理分离模式应用 1概述 毛细管电泳(Caillary Electrophoresis)简称CE，是一类以毛细管为分离通道，以高压直流场为驱动力的新型液相分离分析技术。CE的历史可以追溯到1967年瑞典Hjerten最先提出在直径为3mm的毛细管中做自由溶液的区带电泳(Capillary Zone Electro-phoresis，CZE)。但他没有完全克服传统电泳的弊端[1]。现在所说的毛细管电泳(CE)是由Jorgenson和Lukacs在1981年首先提出，他们使用了75mm的毛细管柱，用荧光检测器对多种组分实现了分离。1984年Terabe将胶束引入毛细管电泳，开创了毛细管电泳的重要分支: 胶束电动毛细管色谱(MEKC)。1987年Hjerten等把传统的等电聚焦过程转移到毛细管内进行。同年，Cohen 发表了毛细管凝胶电泳的工作。近年来，将液相色谱的固定相引入毛细管电泳中，又发展了电色谱，扩大了电泳的应用范围。 毛细管电泳和高效液相色谱（HPLC）一样，同是液相分离技术，因此在很大程度上HPCE与HPLC可以互为补充，但是无论从效率、速度、样品用量和成本来说，毛细管电泳都显示了一定的优势毛细管电泳（C E）除了比其它色谱分离分析方法具有效率更高、速度更快、样品和试剂耗量更少、应用面同样广泛等优点外，其仪器结构也比高效液相色谱（HPLC）简单。C E只需高压直流电源、进样装置、毛细管和检测器。 毛细管电泳具有分析速度快、分离效率高、试验成本低、消耗少、操作简便等特点，因此广泛应用于分子生物学、医学、药学、材料学以及与化学有关的化工、环保、食品、饮料等各个领域[2]。

蛋白质的提取和分离 专题训练

蛋白质的提取和分离专题训练 一．选择题 1．下列有关蛋白质的提取和分离的操作，其中排序正确的是()。 A．样品处理、凝胶色谱操作、SDS－聚丙烯酰胺凝胶电泳 B．样品处理、凝胶色谱操作、纯化C．样品处理、粗分离、纯化、纯度鉴定D．样品处理、纯化、粗分离、纯度鉴定 2．下列对在凝胶柱上加入样品和洗脱的操作不正确的是()。 A．加样前要使柱内凝胶面上的缓冲液下降到与凝胶面平齐 B．让吸管管口沿管壁环绕移动，贴壁加样至色谱柱顶端，不要破坏凝胶面 C．打开下端出口，待样品完全进入凝胶层后直接连接缓冲液洗脱瓶开始洗脱 3．在蛋白质的提取和分离中，下列对样品处理过程的分析正确的是()。 A．洗涤红细胞的目的是去除血浆中的葡萄糖、无机盐 B．洗涤时离心速度过小，时间过短，白细胞等会沉淀，达不到分离的结果 C．洗涤过程选用质量分数0.1%的NaCl溶液(生理盐水) D．透析的目的是去除样品中相对分子质量较小的杂质 4．下列是有关血红蛋白提取和分离的相关操作，其中正确的是()。 A．可采集猪血作为实验材料B．用蒸馏水重复洗涤红细胞 C．血红蛋白释放后应低速短时间离心 D．洗脱液接近色谱柱底端时开始收集流出液 5．蛋白质的分离与纯化技术是蛋白质研究的重要技术。下列叙述不正确的是()。 A．根据蛋白质分子不能透过半透膜的特性，可将样品中各种不同的蛋白质分离 B．根据蛋白质所带电荷性质的差异及分子大小等，可通过电泳分离蛋白质 C．根据蛋白质相对分子质量的大小，可通过凝胶色谱法分离蛋白质 D．根据蛋白质的分子大小、密度不同，可通过离心沉降法分离蛋白质 6．凝胶色谱法分离蛋白质时使用的凝胶是交联葡聚糖凝胶(SephadexG—75)，其中G、75的含义分别是()。 A．G表示凝胶的使用量，75表示加75 g水 B．G表示凝胶的交联程度，75表示需加热75 ℃ C．G表示凝胶的交联程度、膨胀程度及分离范围，75表示每克凝胶膨胀时吸水7.5 g D．G表示凝胶的膨胀体积，75表示每克凝胶膨胀后体积可达75 cm3 7．用凝胶色谱法分离蛋白质时，相对分子质量大的蛋白质()。 A．路程较长，移动速度较慢B．路程较长，移动速度较快 C．路程较短，移动速度较慢D．路程较短，移动速度较快 8．下列关于蛋白质提取和分离实验中样品处理步骤的描述，正确的是()。 A．红细胞的洗涤：加入蒸馏水，缓慢搅拌，低速短时间离心 B．血红蛋白的释放：加入生理盐水和甲苯，置于磁力搅拌器上充分搅拌 C．分离血红蛋白：将搅拌好的混合液离心，过滤后，用分液漏斗分离 D．透析：将血红蛋白溶液装入透析袋，然后置于pH为4.0的磷酸缓冲液中透析12 h 9．下列各项中不属于电泳使样品中各分子分离的原因的是()。 A．分子带电性质的差异B．分子的大小C．分子的形状D．分子的变性温度10．下列有关提取和分离血红蛋白的程序叙述错误的是()。 A．样品的处理就是通过一系列操作收集到血红蛋白溶液 B．通过透析可以去除样品中相对分子质量较大的杂质，此为样品的粗提取 C．可通过凝胶色谱法将相对分子质量大的杂质蛋白除去，即样品的纯化 D．可通过SDS－聚丙烯酰胺凝胶电泳鉴定血红蛋白的纯度 11．下列有关蛋白质提取和分离的叙述中，错误的是()。 A．透析法分离蛋白质的原理是利用蛋白质不能通过半透膜的特性 B．采用透析法使蛋白质与其他小分子化合物分离开来 C．离心沉降法通过控制离心速率使分子大小、密度不同的蛋白质分离 D．蛋白质在电场中可以向与其自身所带电荷相同的电极移动

蛋白质分离纯化的步骤

蛋白质分离纯化的一般程序可分为以下几个步骤： （一）材料的预处理及细胞破碎 分离提纯某一种蛋白质时，首先要把蛋白质从组织或细胞中释放出来并保持原来的天然状态，不丧失活性。所以要采用适当的方法将组织和细胞破碎。常用的破碎组织细胞的方法有： 1. 机械破碎法 这种方法是利用机械力的剪切作用，使细胞破碎。常用设备有，高速组织捣碎机、匀浆器、研钵等。 2. 渗透破碎法 这种方法是在低渗条件使细胞溶胀而破碎。 3. 反复冻融法 生物组织经冻结后，细胞内液结冰膨胀而使细胞胀破。这种方法简单方便，但要注意那些对温度变化敏感的蛋白质不宜采用此法。 4. 超声波法 使用超声波震荡器使细胞膜上所受张力不均而使细胞破碎。 5. 酶法 如用溶菌酶破坏微生物细胞等。 （二）蛋白质的抽提 通常选择适当的缓冲液溶剂把蛋白质提取出来。抽提所用缓冲液的pH、离子强度、组成成分等条件的选择应根据欲制备的蛋白质的性质而定。如膜蛋白的抽提，抽提缓冲液中一般要加入表面活性剂（十二烷基磺酸钠、tritonX-100 等），使膜结构破坏，利于蛋白质与膜分离。在抽提过程中，应注意温度，避免剧烈搅拌等，以防止蛋白质的变性。（三）蛋白质粗制品的获得选用适当的方法将所要的蛋白质与其它杂蛋白分离开来。比较方便的有效方法是根据蛋白质溶解度的差异进行的分离。常用的有下列几种方法： 1.等电点沉淀法不同蛋白质的等电点不同，可用等电点沉淀法使它们相互分离。 2.盐析法 不同蛋白质盐析所需要的盐饱和度不同，所以可通过调节盐浓度将目的蛋白沉淀析出。被盐析沉淀下来的蛋白质仍保持其天然性质，并能再度溶解而不变性。 3.有机溶剂沉淀法 中性有机溶剂如乙醇、丙酮，它们的介电常数比水低。能使大多数球状蛋白质在水溶液中的溶解度降低，进而从溶液中沉淀出来，因此可用来沉淀蛋白质。此外，有机溶剂会破坏蛋白质表面的水化层，促使蛋白质分子变得不稳定而析出。由于有机溶剂会使蛋白质变性，使用该法时，要注意在低温下操作，选择合适的有机溶剂浓度。 （四）样品的进一步分离纯化

蛋白质提取

蛋白质提取与制备蛋白质种类很多，性质上的差异很大，既或是同类蛋白质，因选用材料不同，使用方法差别也很大，且又处于不同的体系中，因此不可能有一个固定的程序适用各类蛋白质的分离。但多数分离工作中的关键部分基本手段还是共同的，大部分蛋白质均可溶于水、稀盐、稀酸或稀碱溶液中，少数与脂类结合的蛋白质溶于乙醇、丙酮及丁醇等有机溶剂中。因此可采用不同溶剂提取、分离及纯化蛋白质和酶。 蛋白质与酶在不同溶剂中溶解度的差异，主要取决于蛋白分子中非极性疏水基团与极性亲水基团的比例，其次取决于这些基团的排列和偶极矩。故分子结构性质是不同蛋白质溶解差异的内因。温度、pH、离子强度等是影响蛋白质溶解度的外界条件。提取蛋白质时常根据这些内外因素综合加以利用。将细胞内蛋白质提取出来。并与其它不需要的物质分开。但动物材料中的蛋白质有些可溶性的形式存在于体液（如血浆、消化硫等）中，可以不必经过提取直接进行分离。蛋白质中的角蛋白、胶原及丝蛋白等不溶性蛋白质，只需要适当的溶剂洗去可溶性的伴随物，如脂类、糖类以及其他可溶性蛋白质，最后剩下的就是不溶性蛋白质。这些蛋白质经细胞破碎后，用水、稀盐酸及缓冲液等适当溶剂，将蛋白质溶解出来，再用离心法除去不溶物，即得粗提取液。水适用于白蛋白类蛋白质的抽提。如果抽提物的pH用适当缓冲液控制时，共稳定性及溶解度均能增加。如球蛋白类能溶于稀盐溶液中，脂蛋白可用 稀的去垢剂溶液如十二烷基硫酸钠、洋地黄皂苷（Digitonin）溶液或有机溶剂来抽提。其它不溶于水的蛋白质通常用稀碱溶液抽提。 蛋白质类别和溶解性质 白蛋白和球蛋白: 溶于水及稀盐、稀酸、稀碱溶液，可被50%饱和度硫酸铵析出。真球蛋白: 一般在等电点时不溶于水，但加入少量的盐、酸、碱则可溶解。 拟球蛋白: 溶于水，可为50%饱和度硫酸铵析出 醇溶蛋白: 溶于70～80%乙醇中，不溶于水及无水乙醇 壳蛋白: 在等电点不溶于水，也不溶于稀盐酸，易溶于稀酸、稀碱溶液 精蛋白: 溶于水和稀酸，易在稀氨水中沉淀 组蛋白: 溶于水和稀酸，易在稀氨水中沉淀 硬蛋白质: 不溶于水、盐、稀酸及稀碱 缀合蛋白(包括磷蛋白、粘蛋白、糖蛋白、核蛋白、脂蛋白、血红蛋白、金属蛋白、黄素蛋白和氮苯蛋白等) : 此类蛋白质溶解性质随蛋白质与非蛋白质结合部分的不同而异，除脂蛋白外，一般可溶于稀酸、稀碱及盐溶液中，脂蛋白如脂肪部分露于外，则脂溶性占优势，如脂肪部分被包围于分子之中，则水溶性占优势。 蛋白质的制备是一项十分细致的工作。涉及物理学、化学和生物学的知识很广。近年来虽然有了不改进，但其主要原理仍不外乎两个方面： 一是利用混合物中几个组分分配率的差别，把它们分配于可用机械方法分离的两个或几个物相中，如盐析、有机溶剂提取、层析和结晶等； 二是将混合物置于单一物相中，通过物理力场的作用使各组分分配于不同区域而达到分离的目的，如电泳、超离心、超滤等。由于蛋白质不能溶化，也不能蒸发，所能分配的物相只限于固相和液相，并在这两相间互相交替进行分离纯化。 制备方法可按照分子大小、形状、带电性质及溶解度等主要因素进行分类。按分子大小和形态分为差速离心、超滤、分子筛及透析等方法；按溶解度分为盐析、溶剂抽提、分配层析、逆流分配及结晶等方法；按电荷差异分为电泳、电渗析、等电点沉淀、离子交换层析及吸附层析等；按生物功能专一性有亲合层析法等。 由于不同生物大分子结构及理化性质不同，分离方法也不一样。即同一类生物大分子由于选用材料不同，使用方法差别也很大。因此很难有一个统一标准的方法对任何蛋白质均可循用。因此实验前应进行充分调查研究，查阅有关文献资料，对欲分离提纯物质的物理、化学及生物学性质先有一定了解，然后

蛋白质的盐析与透析

蛋白质的分离纯化 一、实验目的 1.了解蛋白质的分离纯化方法 2.掌握蛋白质的盐析及透析方法 二、实验原理 在蛋白质溶液中加入一定浓度的中性盐，蛋白质即从溶液中沉淀析出，这种作用称为盐析。盐析法常用的盐类有硫酸铵、硫酸钠等。 蛋白质用盐析法沉淀分离后，需脱盐才能获得纯品，脱盐最常用的方法为透析法。蛋白质在溶液中因其胶体质点直径较大，不能透过半透膜，而无机盐及其它低分子物质可以透过，故利用透析法可以把经盐析法所得的蛋白质提纯，即把蛋白质溶液装入透析袋内，将袋口用线扎紧，然后把它放进蒸馏水或缓冲液中，蛋白质分子量大，不能透过透析袋而被保留在袋内，通过不断更换袋外蒸馏水或缓冲液，直至袋内盐分透析完为止。透析常需较长时间，宜在低温下进行。 三、实验材料和试剂 10%鸡蛋白溶液，含鸡蛋清的氯化钠蛋白溶液，饱和硫酸铵溶液，硫酸铵晶体，1%硝酸银溶液，双缩脲试剂 四、实验步骤 （一）蛋白质盐析 取10%鸡蛋白溶液5ml于试管中，加入等量饱和硫酸铵溶液，微微摇动试管，使溶液混合后静置数分钟，蛋白即析出，如无沉淀可再加少许饱和硫酸铵溶液，观察蛋白质的析出； 取少量沉淀混合物，加水稀释，观察沉淀是否会再溶解。 （二）蛋白质的透析 注入含鸡蛋清的氯化钠蛋白溶液5ml于透析袋中，将袋的开口端用线扎紧，然后悬挂在盛有蒸馏水的烧杯中，使其开口端位于水面之上。 经过10分钟后，自烧杯中取出1ml溶液于试管中，加1%硝酸银溶液一滴，如有白色氯化银沉淀生成，即证明蒸馏水中有Cl-存在。 再自烧杯中取出1ml溶液于另一试管中，加入1ml 10%的氢氧化钠溶液，然后滴加1-2滴1%的硫酸铜溶液，观察有无蓝紫色出现。 每隔20分钟更换蒸馏水一次，经过数小时，则可观察到透析袋内出现轻微混浊，此即为蛋白质沉淀。继续透析至蒸馏水中不再生成氯化银沉淀为止。 实验报告记录透析完毕所需的时间。

蛋白质的分离纯化方法(参考资料)

蛋白质的分离纯化方法 2.1根据分子大小不同进行分离纯化 蛋白质是一种大分子物质,并且不同蛋白质的分子大小不同,因此可以利用一些较简单的方法使蛋白 质和小分子物质分开,并使蛋白质混合物也得到分离。根据蛋白质分子大小不同进行分离的方法主要有透析、超滤、离心和凝胶过滤等。透析和超滤是分离蛋白质时常用的方法。透析是将待分离的混合物放入半透膜制成的透析袋中,再浸入透析液进行分离。超滤是利用离心力或压力强行使水和其它小分子通过半透膜,而蛋白质被截留在半透膜上的过程。这两种方法都可以将蛋白质大分子与以无机盐为主的小分子分开。它们经常和盐析、盐溶方法联合使用,在进行盐析或盐溶后可以利用这两种方法除去引入的无机盐。由于超滤过程中,滤膜表面容易被吸附的蛋白质堵塞,以致超滤速度减慢,截流物质的分子量也越来越小。所以在使用超滤方法时要选择合适的滤膜,也可以选择切向流过滤得到更理想的效果离心也是经常和其它方法联合使用的一种分离蛋白质的方法。当蛋白质和杂质的溶解度不同时可以利用离心的方法将它们分开。例如,在从大米渣中提取蛋白质的实验中,加入纤维素酶和α-淀粉酶进行预处理后,再用离心的方法将有用物质与分解掉的杂质进行初步分离[3]。使蛋白质在具有密度梯度的介质中离心的方法称为密度梯度(区带)离心。常用的密度梯度有蔗糖梯度、聚蔗糖梯度和其它合成材料的密度梯度。可以根据所需密度和渗透压的范围选择合适的密度梯度。密度梯度离心曾用于纯化苏云金芽孢杆菌伴孢晶体蛋白,得到的产品纯度高但产量偏低。蒋辰等[6]通过比较不同密度梯度介质的分离效果,利用溴化钠密度梯度得到了高纯度的苏云金芽孢杆菌伴孢晶体蛋白。凝胶过滤也称凝胶渗透层析,是根据蛋白质分子大小不同分离蛋白质最有效的方法之一。凝胶过滤的原理是当不同蛋白质流经凝胶层析柱时,比凝胶珠孔径大的分子不能进入珠内网状结构,而被排阻在凝胶珠之外,随着溶剂在凝胶珠之间的空隙向下运动并最先流出柱外;反之,比凝胶珠孔径小的分子后流出柱外。目前常用的凝胶有交联葡聚糖凝胶、聚丙烯酰胺凝胶和琼脂糖凝胶等。在甘露糖蛋白提纯的过程中使用凝胶过滤方法可以得到很好的效果,纯度鉴定证明产品为分子量约为32 kDa、成分是多糖∶蛋白质(88∶12)、多糖为甘露糖的单一均匀糖蛋白[1]。凝胶过滤在抗凝血蛋白的提取过程中也被用来除去大多数杂蛋白及小分子的杂质[7]。 2.2 根据溶解度不同进行分离纯化 影响蛋白质溶解度的外部条件有很多,比如溶液的pH值、离子强度、介电常数和温度等。但在同一条件下,不同的蛋白质因其分子结构的不同而有不同的溶解度,根据蛋白质分子结构的特点,适当地改变外部条件,就可以选择性地控制蛋白质混合物中某一成分的溶解度,达到分离纯化蛋白质的目的。常用的方法有等电点沉淀和pH值调节、蛋白质的盐溶和盐析、有机溶剂法、双水相萃取法、反胶团萃取法等。 等电点沉淀和pH值调节是最常用的方法。每种蛋白质都有自己的等电点,而且在等电点时溶解度最

蛋白质的提取与纯化

蛋白质的提取与纯化 一，蛋白质的提取 大部分蛋白质都可溶于水、稀盐、稀酸或碱溶液，少数与脂类结合的蛋白质则溶于乙醇、丙酮、丁醇等有机溶剂中，因些，可采用不同溶剂提取分离和纯化蛋白质及酶。 （一）水溶液提取法 稀盐和缓冲系统的水溶液对蛋白质稳定性好、溶解度大、是提取蛋白质最常用的溶剂，通常用量是原材料体积的1-5倍，提取时需要均匀的搅拌，以利于蛋白质的溶解。提取的温度要视有效成份性质而定。一方面，多数蛋白质的溶解度随着温度的升高而增大，因此，温度高利于溶解，缩短提取时间。但另一方面，温度升高会使蛋白质变性失活，因此，基于这一点考虑提取蛋白质和酶时一般采用低温（5度以下）操作。为了避免蛋白质提以过程中的降解，可加入蛋白水解酶抑制剂（如二异丙基氟磷酸，碘乙酸等）。 下面着重讨论提取液的pH值和盐浓度的选择。 1、pH值 蛋白质，酶是具有等电点的两性电解质，提取液的pH值应选择在偏离等电点两侧的pH 范围内。用稀酸或稀碱提取时，应防止过酸或过碱而引起蛋白质可解离基团发生变化，从而导致蛋白质构象的不可逆变化，一般来说，碱性蛋白质用偏酸性的提取液提取，而酸性蛋白质用偏碱性的提取液。 2、盐浓度 稀浓度可促进蛋白质的溶，称为盐溶作用。同时稀盐溶液因盐离子与蛋白质部分结合，具有保护蛋白质不易变性的优点，因此在提取液中加入少量NaCl等

中性盐，一般以0.15摩尔。升浓度为宜。缓冲液常采用0.02-0.05M磷酸盐和碳酸盐等渗盐溶液。 （二）有机溶剂提取法 一些和脂质结合比较牢固或分子中非极性侧链较多的蛋白质和酶，不溶于水、稀盐溶液、稀酸或稀碱中，可用乙醇、丙酮和丁醇等有机溶剂，它们具的一定的亲水性，还有较强的亲脂性、是理想的提脂蛋白的提取液。但必须在低温下操作。丁醇提取法对提取一些与脂质结合紧密的蛋白质和酶特别优越，一是因为丁醇亲脂性强，特别是溶解磷脂的能力强；二是丁醇兼具亲水性，在溶解度范围内（度为10%，40度为6.6%）不会引起酶的变性失活。另外，丁醇提取法的pH及温度选择范围较广，也适用于动植物及微生物材料。 二、蛋白质的分离纯化 蛋白质的分离纯化方法很多，主要有： （一）根据蛋白质溶解度不同的分离方法 1、蛋白质的盐析 中性盐对蛋白质的溶解度有显著影响，一般在低盐浓度下随着盐浓度升高，蛋白质的溶解度增加，此称盐溶；当盐浓度继续升高时，蛋白质的溶解度不同程度下降并先后析出，这种现象称盐析，将大量盐加到蛋白质溶液中，高浓度的盐离子（如硫酸铵的SO4和NH4)有很强的水化力，可夺取蛋白质分子的水化层，使之“失水”，于是蛋白质胶粒凝结并沉淀析出。盐析时若溶液pH在蛋白质等电点则效果更好。由于各种蛋白质分子颗粒大小、亲水程度不同，故盐析所需的盐浓度也不一样，因此调节混合蛋白质溶液中的中性盐浓度可使各种蛋白质分段沉淀。

盐析法

盐析法综述 摘要：沉淀法是利用沉淀反应，将被测组分转化为难溶物，以沉淀形式从溶液中分离出来，并转化为称量形式，最后称定其重量进行测定的方法。盐析法是其中的一种，盐析法是在中药水提液中,加入无机盐至一定浓度,或达饱和状态,可使某些成分在水中溶解度降低,从而与水溶性大的杂质分离。常作盐析的无机盐有氯化钠、硫酸钠、硫酸镁、硫酸铵等。 关键词:沉淀法；盐析；原理；方法评价；蛋白质盐析 沉淀法 沉淀法是利用沉淀反应，将被测组分转化为难溶物，以沉淀形式从溶液中分离出来，并转化为称量形式，最后称定其重量进行测定的方法。 有机溶剂沉淀法多用于生物小分子、多糖及核酸产品的分离纯化，有时也用于蛋白质沉淀。有机溶剂的沉淀机理是降低水的介电常数，导致具有表面水层的生物大分子脱水，相互聚集，最后析出。等电点沉淀法用于氨基酸、蛋白质及其它两性物质的沉淀。但此法单独应用较少，多与其它方法结合使用。两性电解质分子上的净电荷为零时溶解度最低，不同的两性电解质具有不同的等电点，以此为基础可进行分离。、非离子多聚体沉淀法用于分离生物大分子非离子多聚物是六十年代发展起来的一类重要沉淀剂，最早用于提纯免疫球蛋白、沉淀一些细菌和病毒，近年来逐渐广泛应用于核酸和酶的分离提纯。最常用的是铅盐法，可以用于除去杂质，也可用于沉淀有效成分。沉淀法通常是在溶液状态下将不同化学成分的物质混合,在混合液中加人适当的沉淀剂制备前驱体沉淀物,再将沉淀物进行干燥或锻烧,从而制得相应的粉体颗粒。一般来说，所有固体溶质都可以在溶液中加入中性盐而沉淀析出，这一过程叫盐析。在生化制备中,许多物质都可以用盐析法进行沉淀分离，如蛋白质、多肽、多糖、核酸等，其中以蛋白质沉淀最为常见，特别是在粗提阶段。 对沉淀形式的要求 （1）沉淀的溶解度要小，以保证被测组分能沉淀完全。 （2）沉淀要纯净，不应带入沉淀剂和其他杂质。 （3）沉淀易于过滤和洗涤，以便于操作和提高沉淀的纯度。 （4）沉淀易于转化为称量形式。 盐析法 胶体的盐析 胶体的盐析是加盐而使胶粒的溶解度降低，形成沉底析出的

中图版高中生物选修1 6.1蛋白质的提取和分离_教案设计1

蛋白质的提取和分离 【教学目标】 1．知道蛋白质分离和提取技术的基本原理。 2．能进行血清蛋白的提取和分离。 3．尝试提取和检测牛奶中的酪蛋白。 4．尝试卵清蛋白的分离。 【教学重难点】 1．知道蛋白质分离和提取技术的基本原理。 2．能进行血清蛋白的提取和分离。 【教学过程】 一、蛋白质分离提纯技术 1．将生物体内的蛋白质提取出来并加以分离，就可以得到高纯度的、甚至是单一种类的蛋白质。 2．蛋白质分离提纯技术包括离心技术、层析技术和电泳技术等。其中，电泳技术最为快捷灵敏、简便易行。 二、电泳技术及分离蛋白质的原理 1．电泳现象：带电粒子在电场中可以向与其自身所带电荷相反的电极方向移动。电泳常用的支持物是聚丙烯酰胺凝胶，由此而来的技术称为聚丙烯酰胺凝胶电泳。 2．电泳技术分离蛋白质的原理： （1）蛋白质含有游离的氨基和羧基，是两性电解质，在一定pH条件下带有电荷。 （2）蛋白质所带的电荷数越多，移动得越快；电荷数相同时，分子量越小，则移动越快。 （3）不同蛋白质的混合溶液，在经过同一个电场电泳后，会在凝胶上形成不同的带纹。每一种带纹可能就是一种蛋白质。将这些带纹一个一个地剪下来，分别予以洗脱，然后对洗脱液中的蛋白质做进一步的分离。如果待测洗脱液不能再分离，则这个带纹中很可能含有一种单纯蛋白质。如果待测洗脱液还能再分离，则这个带纹中含有多种蛋白质，需要作进一步分离，直至提纯。 三、血清蛋白的提取和分离 1．新鲜血液在体外凝固后，会析出清亮的淡黄色液体——血清。血清中含有丙种球蛋白、

凝集素等多种蛋白质。 2．过程： （1）点样：取新制血清与蔗糖溶液及溴酚蓝指示剂等体积混匀后，小心加到电泳样品槽的胶面上。 （2）电泳。 （3）染色：取出凝胶，放入质量分数为0.05%的考马斯亮蓝R250染色液中。染色与固定同时进行，使染色液没过胶板，染色30min。 （4）脱色：用体积分数为7%的醋酸溶液浸泡漂洗数次，每隔1~2h更换脱色液，直至底色脱净，背景清晰。 （5）制干胶板：将已脱色的凝胶板放在保存液中浸泡3~4h，然后放在两层透气玻璃纸中间，自然干燥即可获得血清蛋白的电泳谱带。 四、其他蛋白质的提取与分离 1．牛奶中酪蛋白的提取与检测： 当pH=4.8时，酪蛋白的溶解度降低，并析出沉淀。用酒精除去酪蛋白沉淀中的脂肪后，即得到纯的酪蛋白。 酪蛋白中含有酪氨酸，能与米伦试剂起颜色反应，首先生成白色沉淀，加热后变成红色。 2．卵清蛋白质的分离： 选取鸡、鸭、鹅、鹌鹑等动物的新鲜卵清做实验材料。采用电泳法分离卵清蛋白质。 自主思考： 可否利用蛋白质的提取与分离技术快速诊断早期癌变？ 提示：可以。只需从早期患者的尿液、血液或细胞裂解液中提取少量蛋白质样品，采用蛋白质指纹图谱技术，就可以更加快速、简便、准确地对细胞癌变做出诊断。 探究一：蛋白质的分离原理 问题导引： 为年老多病的人注射丙种球蛋白，可以在一定程度上增强其身体的抵抗力。在动物体内，丙种球蛋白和许多蛋白质混在一起。要获得纯净的丙种球蛋白，就必须进行提取和分离。你能提供分离蛋白质的思路吗？ 提示：可根据蛋白质各种特性的差异，如分子的大小、所带电荷的性质和多少等来分离不同种类的蛋白质。 名师精讲： 蛋白质的分离方法：

蛋白质的提取与分离

蛋白质提取与制备具体操作方法 1、原料的选择 早年为了研究的方便，尽量寻找含某种蛋白质丰富的器官从中提取蛋白质。但至目前经常遇到的多是含量低的器官或组织且量也很小，如下丘脑、松果体、细胞膜或内膜等原材料，因而对提取要求更复杂一些。 原料的选择主要依据实验目的定。从工业生产角度考虑，注意选含量高、来源丰富及成 本低的原料。尽量要新鲜原料。但有时这几方面不同时具备。含量丰富但来源困难，或含量 来源均理想，但分离纯化操作繁琐，反而不如含量略低些易于获得纯品者。一般要注意种属 的关系，如鲣的心肌细胞色素C较马的易结晶，马的血红蛋白较牛的易结晶。要事前调查 制备的难易情况。若利用蛋白质的活性，对原料的种属应几乎无影响。如利用胰蛋白酶水解 蛋白质的活性，用猪或牛胰脏均可。但若研究蛋白质自身的性质及结构时，原料的来源种属 必须一定。研究由于病态引起的特殊蛋白质（本斯.琼斯氏蛋白、贫血血红蛋白）时，不但 使用种属一定的原料，而且要取自同一个体的原料。可能时尽量用全年均可采到的原料。对 动物生理状态间的差异（如饥饿时脂肪和糖类相对减少），采收期及产地等因素也要注意。 2、前处理 a、细胞的破碎 材料选定通常要进行处理。要剔除结缔组织及脂肪组织。如不能立即进行实验，则应冷 冻保存。除了提取及胞细外成分，对细胞内及多细胞生物组织中的蛋白质的分离提取均须先 将细胞破碎，使其充分释放到溶液中。不同生物体或同一生物体不同的组织，其细胞破坏难 易不一，使用方法也不完全相同。如动物胰、肝、脑组织一般较柔软，作普通匀浆器磨研即 可，肌肉及心组织较韧，需预先绞碎再制成匀桨。 ⑴机械方法 主要通过机械切力的作用使组织细胞破坏。常用器械有：①高速组织捣碎机（转速可达 10000rpm，具高速转动的锋利的刀片），宜用于动物内脏组织的破碎；②玻璃匀浆器（用两 个磨砂面相互摩擦，将细胞磨碎），适用于少量材料，也可用不锈钢或硬质塑料等，两面间 隔只有十分之几毫米，对细胞破碎程度较高速捣碎机高，机械切力对分子破坏较小。小量的 也可用乳钵与适当的缓冲剂磨碎提取，也可加氧化铝、石英砂及玻璃粉磨细。但在磨细时局 部往往生热导致变性或pH显著变化，尤其用玻璃粉和氧化铝时。磨细剂的吸附也可导致损 失。 ⑵物理方法 主要通过各种物理因素的作用，使组织细胞破碎的方法。

分离纯化蛋白质的方法及原理

（二）利用溶解度差别 影响蛋白质溶解度的外部因素有：1、溶液的pH；2、离子强度；3、介电常数；4、温度。但在同一的特定外部条件下，不同蛋白质具有不同的溶解度。 1、等电点沉淀：原理：蛋白质处于等电点时，其净电荷为零，由于相邻蛋白质分子之间没有静电斥力而趋于聚集沉淀。因此在其他条件相同时，他的溶解度达到最低点。在等电点之上或者之下时，蛋白质分子携带同种符号的净电荷而互相排斥，阻止了单个分子聚集成沉淀，因此溶解度较大。不同蛋白质具有不同的等电点，利用蛋白质在等电点时的溶解度最低的原理，可以把蛋白质混合物分开。当pH被调到蛋白质混合物中其中一种蛋白质的等电点时，这种蛋白质大部分和全部被沉淀下来，那些等电点高于或低于该pH的蛋白质则仍留在溶液中。这样沉淀出来的蛋白质保持着天然的构象，能重新溶解于适当的pH和一定浓度的盐溶液中。 5、盐析与盐溶：原理：低浓度时，中性盐可以增加蛋白质溶解度这种现象称为盐溶.盐溶作用主要是由于蛋白质分子吸附某种盐类离子后，带电层使蛋白质分子彼此排斥，而蛋白质与水分子之间的相互作用却加强，因而溶解度增高。球蛋白溶液在透析过程中往往沉淀析出，这就是因为透析除去了盐类离子，使蛋白质分子之间的相互吸引增加，引起蛋白质分子的凝集并沉淀。当溶液的离子强度增加到一定程度时，蛋白质溶解程度开始下降。当离子强度增加到足够高时，例如饱和或半饱和程度，很多蛋白质可以从水中沉淀出来，这种现象称为盐析。盐析作用主要是由于大量中性盐的加入使水的活度降低，原来溶液中的大部分甚至全部的自由水转变为盐离子的水化水。此时那些被迫与蛋白质表面的疏水集团接触并掩盖他们的水分子成为下一步最自由的可利用的水分子，因此被移去以溶剂化盐离子，留下暴露出来的疏水基团。蛋白质疏水表面进一步暴露，由于疏水作用蛋白质聚集而沉淀。 盐析沉淀的蛋白质保持着他的天然构象，能再溶解。盐析的中性盐以硫酸铵为最佳，在水中的溶解度很高，而溶解度的温度系数较低。 3、有机溶剂分级分离法：与水互溶的有机溶剂（甲醇、乙醇和丙酮等）能使蛋白质在水中的溶解度显著降低。在室温下有机溶剂会引起蛋白质变性，如果预先将有机溶剂冷却到-40°C以下，然后在不断搅拌下逐滴加入有机溶剂，以防局部浓度过高，那么变性可以得到很大程度缓解。蛋白质在有机溶剂中的溶解度也随温度、pH和离子强度而变化。在一定温度、pH和离子强度条件下，引起蛋白质沉淀的有机溶剂的浓度不同，因此控制有机溶剂浓度也可以分

蛋白质分离与纯化教学设计课题

蛋白质分离与纯化教学设计 一、教学背景分析 【教材分析】 “蛋白质的分离与纯化”实验是《高中生物》选修1生物技术实践 5.3血红蛋白的提取与分离中的容。本节课的主要容包括蛋白质的提取、分离纯化等基本知识，主要要求学生掌握凝胶电泳的实验原理以及操作方法。“血红蛋白分离与纯化”实验不仅是学习血红蛋白的提取、分离纯化方法，而且也是进一步掌握蛋白质的组成、结构和功能的基础。 【学情分析】 到目前为止，学生已经学习了蛋白质的相关知识，对蛋白质有了一定的了解，“蛋白质的分离与纯化”实验目的是使学生体验从复杂细胞混合物体系中提取和纯化生物大分子的基本原理、过程和方法，虽然操作难度较大，但原理清晰，动手机会较多，学习兴趣很高。学生有必修“生命活动的主要承担者——蛋白质”的基础，在一定程度上掌握了蛋白质的组成、结构和功能等基础知识，学生在进行实验前还是能大概了解影响蛋白质分离纯化的因素的，再者经过老师的指导，实验能取得良好的结果的。 二、教学目标 【知识目标】 1.了解从血液中提取蛋白质的原理与方法。 2.说出凝胶电泳的基本原理与方法。 【能力目标】 运用凝胶电泳对蛋白质进行分离纯化。 【情感态度与价值观目标】 1.培养学生科学实验的观点。 2.初步形成科学的思维方式，发展科学素养和人文精神。 三、教学重难点

【教学重点】 从血液中提取蛋白质；凝胶电泳分离纯化蛋白质。 【教学难点】 样品预处理，色谱柱的装柱，纯化分离操作。 四、实验实施准备 【教师准备】 1.分组。学生按学科能力的强中弱平均分组，各组尽量平衡，各组自行分工，并由实验员统一安排实验过程。 2.实验材料：血液 仪器：试管、胶头滴管、烧杯、玻璃棒、离心机、研磨器、透析袋、电泳仪等。 试剂：20mmol/L磷酸缓冲液（pH为8．6）、蒸馏水、聚丙烯酸铵、生理盐水、5％醋酸水溶液等。 【学生准备】 1.预习实验“蛋白质分离纯化”，了解蛋白质的相关信息。 2.进行分组。 五、教学方法 【教法】分析评价法、任务驱动法、直观演示法 【学法】自主学习法、合作交流法 六、教学媒体 黑板、多媒体 七、课时安排 两个课时（80min） 一个课时用来讲述理论部分知识：样品处理与色谱柱分离纯化蛋白质的原理与方法； 另一课时用来进行实验。

【实验操作】蛋白质的提取和分离实验操作

优选精品资源欢迎下载选用 实验操作 蛋白质的提取和分离一般分为四步:样品处理、粗分离、纯化和纯度鉴定。 (1)样品处理 ①红细胞的洗涤洗涤红细胞的目的:去除杂蛋白，以利于后续步骤的分离纯化。采集的血样要及时分离红细胞，分离时采用低速短时间离心，如500r/min离心2min，然后用胶头吸管吸出上层透明的黄色血浆，将下层暗红色的红细胞液体倒入烧杯，再加入五倍体积的生理盐水，缓慢搅拌10min，低速短时间离心，如此重复洗涤三次，直至上清液中没有黄色，表明红细胞已洗涤干净。洗涤次数、离心速度与离心时间十分重要。洗涤次数过少，无法除去血浆蛋白;离心速度过高和时间过长会使白细胞等一同沉淀，达不到分离的效果。 ②血红蛋白的释放将洗涤好的红细胞倒人烧杯中，加蒸馏水到原血液的体积，再加40%体积的甲苯，置于磁力搅拌器上充分搅拌10min。蒸馏水和甲苯作用:使红细胞破裂释放出血红蛋白。 ③分离血红蛋白溶液将搅拌好的混合液转移到离心管中，以20**r/min的速度离心10min后，可以明显看到试管中的液体分为4层。第1层为无色透明的甲苯层，第2层为白色薄层固体，是脂溶性物质的沉淀层，第3层是红色透明液体，这是血红蛋白的水溶液，第4层是其他杂质的暗红色沉淀物。将试管中的液体用滤纸过滤，除去脂溶性沉淀层，于分液漏斗中静置片刻后，分出下层的红色透明液体。 ④透析取lmL的血红蛋白溶液装入透析袋中，将透析袋故人盛有300mL的物质的量浓度为20mmol/L的磷酸缓冲液中(pH为7.0)，透析12h。 (2)凝胶色谱操作 ①凝胶色谱柱的制作 ②凝胶色谱柱的装填将色谱柱垂直固定在支架上。计算所用凝胶量，并称量。凝胶用蒸馏水充分溶胀后，配成凝胶悬浮液，在与色谱柱下端连接的尼龙臂打开的情况下，一次性缓慢倒入色谱柱内，装填时可轻轻敲动色谱柱，使凝胶装填均匀。色谱柱内不能有气泡存在，一旦发现有气泡，必须重装。装填完后，立即连接缓冲液洗脱瓶，在约50cm高的操作压下，用300ml的物质的量浓度为20mmol/L的磷酸缓冲液充分洗涤平衡凝胶12h，使凝胶装填紧密。 ③样品的加入和洗脱打开色谱柱下端的流出口。使柱内凝胶面上的缓冲液缓慢下降到与凝胶面平齐，关闭出口。用吸管小心地将lmL透析后的样品加到色谱柱的顶端，加样时使吸管管口沿管壁环绕移动，注意不要破坏凝胶面。加样后，打开下端出口，使样品渗入凝胶床内。等样品完全进入凝胶层后，关闭下端出口。小心加入物质的量浓度为20mmol/L 的磷酸缓冲液(pH为7.0)到适当高度，连接缓冲液洗脱瓶，打开下端出口，进行洗脱。待红色的蛋白质接近色谱柱底端时，用试管收集流出液，每5mL收集一管，连续收集。

提取蛋白的常规方法

1、原料的选择 早年为了研究的方便，尽量寻找含某种蛋白质丰富的器官从中提取蛋白质。但至目前经 常遇到的多是含量低的器官或组织且量也很小，如下丘脑、松果体、细胞膜或内膜等原材料， - 105 - 蛋白质提取与制备Protein Extraction and Preparation 因而对提取要求更复杂一些。 原料的选择主要依据实验目的定。从工业生产角度考虑，注意选含量高、来源丰富及成 本低的原料。尽量要新鲜原料。但有时这几方面不同时具备。含量丰富但来源困难，或含量 来源均理想，但分离纯化操作繁琐，反而不如含量略低些易于获得纯品者。一般要注意种属 的关系，如鲣的心肌细胞色素C 较马的易结晶，马的血红蛋白较牛的易结晶。要事前调查 制备的难易情况。若利用蛋白质的活性，对原料的种属应几乎无影响。如利用胰蛋白酶水解 蛋白质的活性，用猪或牛胰脏均可。但若研究蛋白质自身的性质及结构时，原料的来源种属 必须一定。研究由于病态引起的特殊蛋白质（本斯.琼斯氏蛋白、贫血血红蛋白）时，不但 使用种属一定的原料，而且要取自同一个体的原料。可能时尽量用全年均可采到的原料。对 动物生理状态间的差异（如饥饿时脂肪和糖类相对减少），采收期及产地等因素也要注意。 2、前处理 a、细胞的破碎 材料选定通常要进行处理。要剔除结缔组织及脂肪组织。如不能立即进行实验，则应冷 冻保存。除了提取及胞细外成分，对细胞内及多细胞生物组织中的蛋白质的分离提取均须先 将细胞破碎，使其充分释放到溶液中。不同生物体或同一生物体不同的组织，其细胞破坏难 易不一，使用方法也不完全相同。如动物胰、肝、脑组织一般较柔软，作普通匀浆器磨研即 可，肌肉及心组织较韧，需预先绞碎再制成匀桨。 ⑴机械方法 主要通过机械切力的作用使组织细胞破坏。常用器械有：①高速组织捣碎机（转速可达 10000rpm，具高速转动的锋利的刀片），宜用于动物内脏组织的破碎；②玻璃匀浆器（用两 个磨砂面相互摩擦，将细胞磨碎），适用于少量材料，也可用不锈钢或硬质塑料等，两面间

高中生物蛋白质和DNA技术第1节蛋白质的提取和分离教案中图版

第一节蛋白质的提取和分离 一、蛋白质分离提纯技术 常用的蛋白质分离提纯技术包括离心技术、层析技术和电泳技术等。其中电泳技术最为快捷灵敏、简便易行。 二、电泳技术分离蛋白质的原理 1．蛋白质中含有游离的氨基和羧基，是两性电解质，在一定pH条件下带有电荷。 2．蛋白质在电场中可以向与其自身所带电荷相反的电极方向移动。 3．蛋白质所带的电荷数越多，移动得越快；电荷数相同时，分子量越小，移动越快。 三、结果分析 不同蛋白质的混合溶液经过同一电场电泳后，会在凝胶上形成不同的带纹，每种带纹可能就是一种蛋白质。 四、“血清蛋白的提取和分离”活动程序 1．点样：取新制血清5微升、质量浓度为0.4克/毫升的蔗糖溶液和质量分数为0.1%的溴酚蓝指示剂等体积混匀后，用微量加样器吸取5 μL样品加到电泳样品槽的胶面上。 2．电泳。 3．染色：用质量分数为0.05%的考马斯亮蓝R250染色液对凝胶进行染色。 4．脱色：用体积分数为7%的醋酸溶液对凝胶板进行浸泡漂洗，至底色脱净。 5．制干胶板：将已脱色的凝胶板放在保存液中浸泡3 h～4 h后，放在两层透气的玻璃纸中间自然干燥。 预习完成后，请把你认为难以解决的问题记录在下面的表格中 问题1 问题2 问题3 问题4

一、蛋白质分离提纯的方法 1．蛋白质分离的基本过程 破碎细胞―→抽提(粗制品)―→纯化蛋白质 2．破碎细胞的方法 在分离与纯化蛋白质之前，必须采用适当的方法使蛋白质呈溶解状态释放出来：通常先将生物组织进行机械破碎，再根据细胞的特点，选择不同的破碎细胞方法(常用的有研磨法、超声波法、酶解法)。 3．抽提 细胞破碎后，各种蛋白质被释放出来，再根据蛋白质的不同性质，选择不同的溶剂进行抽提。 4．纯化 (1)原理：纯化主要是指根据蛋白质之间以及蛋白质与其他物质之间在分子大小、溶解度大小、所带电荷的多少、吸附性质等方面存在的差异进行的。 (2)方法：①透析法——分子大小 ②离心沉淀法——分子大小、密度大小 ③电泳——所带电荷多少 二、“血清蛋白质的分离”实验中应注意的问题 1．由于制备凝胶的丙烯酰胺和双丙烯酰胺具有很强的神经毒性，并且容易被皮肤吸收，因此操作必须在通风橱内或通风处进行。 2.将电泳凝胶片放在考马斯亮蓝R250染色液中染色30 min后，多次更换脱色液至背景清晰。脱色后，可将凝胶浸于保存液中，长期封装在塑料袋内使其不会降低染色强度。为保存永久性记录，可对凝胶进行拍照，或将凝胶干燥成胶片。 电泳的原理及应用 如图为某些氨基酸在pH为6时进行电泳的结果，请回答：

盐析法沉淀蛋白质的原理

盐析法沉淀蛋白质的原理 1 中性盐沉淀（盐析法） 在溶液中加入中性盐使生物大分子沉淀析出的过程称为“盐析”。除了蛋白质和酶以外，多肽、多糖和核酸等都可以用盐析法进行沉淀分离。 盐析法应用最广的还是在蛋白质领域，已有八十多年的历史，其突出的优点是： ①成本低，不需要特别昂贵的设备。 ②操作简单、安全。 ③对许多生物活性物质具有稳定作用。 ⑴中性盐沉淀蛋白质的基本原理 蛋白质和酶均易溶于水，因为该分子的－COOH、－NH2和－OH都是亲水基团，这些基团与极性水分子相互作用形成水化层，包围于蛋白质分子周围形成1nm～100nm颗粒的亲水胶体，削弱了蛋白质分子之间的作用力，蛋白质分子表面极性基团越多，水化层越厚，蛋白质分子与溶剂分子之间的亲和力越大，因而溶解度也越大。亲水胶体在水中的稳定因素有两个：即电荷和水膜。因为中性盐的亲水性大于蛋白质和酶分子的亲水性，所以加入大量中性盐后，夺走了水分子，破坏了水膜，暴露出疏水区域，同时又中和了电荷，破坏了亲水胶体，蛋白质分子即形成沉淀。

⑵中性盐的选择 常用的中性盐中最重要的是（NH4）2SO4，因为它与其他常用盐类相比有十分突出的优点： 1) 溶解度大：尤其是在低温时仍有相当高的溶解度，这是其他盐类所不具备的。由于酶和各种蛋白质通常是在低温下稳定，因而盐析操作也要求在低温下（0～4℃）进行。 2) 分离效果好：有的提取液加入适量硫酸铵 盐析，一步就可以除去75％的杂蛋白，纯 度提高了四倍。 3) 不易引起变性，有稳定酶与蛋白质结构的 作用。有的酶或蛋白质用2～3mol/L浓度的 （NH4）2SO4保存可达数年之久。 4) 价格便宜，废液不污染环境。 ⑶盐析的操作方法 最常用的是固体硫酸铵加入法。将其研成细粉，在搅拌下缓慢均匀少量多次地加入，接近计划饱和度时，加盐的速度更要慢一些，尽量避免局部硫酸铵浓度过大而造成不应有的蛋白质沉淀。盐析后要在冰浴中放置一段时间，待沉淀完全后再离心与过滤。 在低浓度硫酸铵中盐析可采用离心分离，高浓度硫酸铵常用过滤方法。