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ProteoExtract? Transmembrane Protein Extraction Kit Table of Contents

About the Kits (2)

Description 2

Components 2

Storage 2

Equipment and materials required but not supplied 3

ProteoExtract?Transmembrane Protein Extraction Protocol (3)

Extraction of membrane proteins from adherent cultured cells 3

Extraction of membrane proteins from suspension cells or frozen cell pellets 4

Extraction of membrane proteins from tissue 6

Frequently Asked Questions (8)

Appendix (8)

Example extractions 8

Examples of total protein yields using TM-PEK 9

? 2009 EMD Chemicals Inc., an affiliate of Merck KGaA, Darmstadt, Germany. All rights reserved. The Novagen? logo and Novagen? name are registered trademarks of EMD Chemicals Inc. in the United States and in certain other jurisdictions. ProteoExtract? is a registered trademark of Merck KGaA, Darmstadt, Germany. TRITON? is a registered trademark of Dow Chemical Company.

About the Kits

ProteoExtract? Transmembrane Protein Extraction Kit 1 kit 71772-3

Description

The ProteoExtract? Transmembrane Protein Extraction Kit (TM-PEK) uses a novel, detergent-free

chemistry for extraction of peripherally-associated and integral membrane proteins, such as

G-Protein Coupled Receptors (GPCRs), from mammalian cells and tissues. The membrane protein fraction

is directly compatible with enzyme assays, native and denaturing gel electrophoresis, Western blotting,

immunoprecipitation, and (following in-gel tryptic digestion) mass spectrometry.

The TM-PEK method comprises a two-step protocol for the enrichment of transmembrane (TM) proteins. In

the first step, cells or homogenized tissues are permeablized using Extraction Buffer 1 and their soluble

(cytoplasmic) fraction separated from the insoluble (membrane) fraction by centrifugation. In the second

step, membrane proteins are extracted from the lipid bilayer using one of two novel extraction buffers.

These buffers, Extraction Buffer 2A and Extraction Buffer 2B, are prepared by diluting TM-PEK Reagent A

or TM-PEK Reagent B into Extraction Buffer 2. The optimal extraction reagent must be determined

empirically, as it will depend on characteristics of the protein(s) of interest and intended downstream

applications. Extraction Buffer 2A is a very mild extraction agent, allowing for recovery of fragile protein

complexes. Extraction Buffer 2B, by comparison, is a highly efficient extraction agent and facilitates

recovery of difficult-to-extract transmembrane proteins, including those with multiple transmembrane

segments.

If using the TM-PEK kit for the first time, prepare duplicate samples. Extract the first set of replicates with

TM-PEK Reagent A, diluted 2-fold with Extraction Buffer 2. Extract the second set of replicates with TM-

PEK Reagent B, diluted 2-fold with Extraction Buffer 2. Extraction conditions can be optimized further by

varying the dilution range of each reagent into Extraction Buffer, from undiluted to a 10-fold dilution.

Unlike alternative membrane protein extraction methods, the TM-PEK kit does not require sonication,

rigorous vortexing, time-consuming ultracentrifugation, or incubation at elevated temperatures. The absence

of such harsh treatments minimizes potential damage or changes to the target protein.

Each TM-PEK kit provides sufficient reagents to process a total of 40 samples (20 samples using Extraction

Buffer 2A at the recommended dilution, and 20 samples using Extraction Buffer 2B at the recommended

dilution). Each sample may be comprised of 1–5 x 107 cultured cells or 25–50 mg of tissue

Components

?????40 ml Extraction Buffer 1

50 ml Extraction Buffer 2

2.0 ml TM-PEK Reagent A

2.0 ml TM-PEK Reagent B

0.4 ml Protease Inhibitor Cocktail Set III

Storage

Store all components at 4°C for up to 6 months. For prolonged storage, dispense the components in working aliquots and store at –20°C. Before performing extractions, thaw all kit components at room temperature and mix completely. Avoid repeated freezing and thawing.

Note that the protease inhibitors are provided in DMSO and must be kept at room temperature during the extraction procedure to prevent freezing.

Equipment and materials required but not supplied

?

????Rocking platform or elliptical mixer (required at room temperature and 4°C; the device can be transferred from 4°C to room temperature during the course of the experiment)

Homogenizer (e.g., Dounce or Potter-Elvehjem) or mortar and pestle (for tissue samples)

Refrigerated centrifuge with rotor accommodating a 15 ml and/or a 50 ml tube

Refrigerated centrifuge with rotor accommodating a 2 ml tube and generating 16,000 × g

Phosphate Buffered Saline (PBS) (137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4,

pH 7.4)

ProteoExtract?Transmembrane Protein Extraction Protocol

Table 1. Buffer volumes required for membrane protein extraction from cultured cells or tissue.

Source Material Cultured cells* Fresh or frozen tissue

Amount 1.0 5.0 x 102550 mg 100200 mg 2001000 mg

Extraction Buffer 1 (ml) 1.0 1.0 1.0 2.0

Extraction Buffer 2A or 2B (ml) 0.2 0.2

0.5

1.0

Protease Inhibitor (μl) 5 5 20 40

*Adherent, suspension, or frozen pellet

Extraction of membrane proteins from adherent cultured cells

Considerations Before You Begin

The following protocol is optimized for extraction of membrane proteins from adherent cells grown in one

T-75 culture flask. Cells should be of high viability (>90%) and 70–90% confluent

(1.0–2.0 x 107 cells). Different cell types yield considerably different amounts of protein in the membrane protein fraction (see Table 2 on p 9 in Appendix). If low yield is anticipated, the total number of cells can be increased to 5.0 x 107 without increasing reagent volumes (Table 1). For membrane protein extraction from larger cell numbers, we recommend performing replicate extractions from aliquots of 1.0–5.0 x 107 cells. Alternatively, buffer volumes may be scaled up appropriately.

The kit is supplied with two transmembrane solubilization agents, TM-PEK Reagents A and B

(see Description on p 2). Depending on the unique characteristics of the target membrane protein, Extraction Buffer 2A or Extraction Buffer 2B may prove to be more efficient. As a starting point, we recommend testing both membrane extraction buffers. If performing trial extractions using both reagents, carry out Steps 2–9 in parallel on each of two sets of sample replicates.

Extraction Buffer 2A is a mild extraction reagent which can facilitate recovery of protein complexes. Thus, protein yields obtained using Extraction Buffer 2A may be lower than those obtained using Extraction Buffer 2B (see Table 2 on p 9). Extracts can be concentrated with a Vivaspin 2 or Vivaspin 500 Ultrafiltration Centrifugal Device.

Large volumes of TM-PEK 2B reagent can interfere with protein migration in SDS-PAGE. If high resolution is desired, Extraction Buffer 2B samples for SDS-PAGE may be prepared in 2X SDS sample buffer. Alternatively, employ a buffer exchange step.

For most transmembrane proteins, a 2-fold dilution of TM-PEK Reagent A or B in Extraction Buffer 2 results in efficient extraction. For some transmembrane proteins, extraction efficiency may be improved by using TM-PEK Reagent A or B at a different concentration, from undiluted to a 10-fold dilution in Extraction Buffer 2.

Protocol

1.Prepare Extraction Buffer(s) 2A and/or 2B by diluting the appropriate TM-PEK Reagent 2-fold with

Extraction Buffer 2. For membrane protein extraction from 1.0–5.0 x 107 cells, 0.2 ml of Extraction

Buffer 2A or 0.2 ml of Extraction Buffer 2B is required.

- To prepare 0.2 ml of Extraction Buffer 2A, mix 0.1 ml Extraction Buffer 2 and 0.1 ml

TM-PEK Reagent A.

- To prepare 0.2 ml of Extraction Buffer 2B, mix 0.1 ml Extraction Buffer 2 and 0.1 ml

TM-PEK Reagent B.

Notes: We recommend testing both Extraction Buffer 2A and Extraction Buffer 2B to determine which is optimal for your protein of interest.

Samples prepared with Extraction Buffer 2A may require concentration, depending on

downstream application.

High levels of Reagent B can interfere with protein migration on SDS-PAGE. Buffer exchange or

dilution in sample buffer may be required.

A 2-fold dilution of TM-PEK Reagent A or

B in Extraction Buffer 2 results in an efficient extraction

of most proteins, but reagent dilutions may be optimized (from undiluted to a 10-fold dilution).

2.Discard medium from the culture flask.

3.Wash cells two times with PBS at 4°C.

4.Add 3 ml PBS to the culture vessel. Using a cell scraper or a rubber policeman, free cells from the

culture vessel. Transfer cells to a 15 ml conical tube.

5.Centrifuge cells at 1000 × g for 5 min at 4°C. (Alternatively, collect cells by centrifuging at 500 × g for

10 min at 4°C.)

6.Resuspend cells in 1 ml Extraction Buffer 1 + 5 μl Protease Inhibitor Cocktail Set III.

7.Incubate 10 min at 4°C with gentle agitation to avoid formation of cell clumps.

8.Centrifuge at 1000 × g for 5 min at 4°C.

9.Carefully remove supernatant. Store on ice. This is referred to as ‘cytosolic (soluble)’ protein fraction.

10.Resuspend pellet in 0.2 ml Extraction Buffer 2A + 5 μl of Protease Inhibitor Cocktail Set III

or 0.2 ml Extraction Buffer 2B + 5 μl of Protease Inhibitor Cocktail Set III.

11.Incubate for 45 min at room temperature with gentle agitation.

Note: The length and temperature of the incubation at Step 11 can be varied to improve extraction efficiency or to preserve target protein activity. Increasing the incubation time (up to 120 min) can

increase the protein recovery, but may result in decreased target protein activity. Conversely,

incubation at low temperature (4°C) may better preserve activity, but may lower the extraction

efficiency.

12.Centrifuge at 16,000 × g for 15 min at 4°C.

13.Transfer the supernatant, which is enriched in integral membrane proteins, to a fresh tube.

Note: Store cytosolic and membrane fractions on ice if they will be analyzed on the same day. For long-term storage, dispense into aliquots and store at –20°C.

14.Determine total protein concentration of the cytosolic and membrane protein fractions with the BCA

assay.

Note: For some cell types, total protein concentration of the membrane protein fraction (Step 13 above) will be >1.0 mg/ml. A two- to four-fold dilution in sterile, deionized water may be required to bring

the concentration of these samples within the linear region of the BCA standard curve.

Extraction of membrane proteins from suspension cells or frozen cell pellets

Considerations Before You Begin

The following protocol is optimized for extraction of membrane proteins from 1.0–2.0 x 107 cells cultured in

suspension. Cells should be of high viability (>90%). Different cell types yield considerably different

amounts of protein in the membrane protein fraction (see Table 2 on p 9 in Appendix). If low yield is

anticipated, increase the total number of cells to 5.0 x 107 without increasing reagent volumes (see Table 1

on p 3). For extracting membrane proteins from larger cell numbers, perform replicate extractions from

aliquots of 1.0–5.0 x 107 cells. Alternatively, scale up buffer volumes.

The TM-PEK kit is also compatible with frozen cell pellets (1.0–5.0 x 107 cells per extraction). Cells should

be washed a minimum of two times with an appropriate buffer (e.g., PBS) prior to freezing in liquid

nitrogen. If using frozen cell pellets, begin at Step 7 below.

The kit is supplied with two transmembrane solubilization agents, TM-PEK Reagents A and B

(see Description on p 2). Depending on the unique characteristics of the target membrane protein, Extraction

Buffer 2A or Extraction Buffer 2B may prove to be more efficient. As a starting point, we recommend

testing both membrane extraction buffers. If performing trial extractions using both reagents, carry out Steps

2–9 in parallel on each of two sets of sample replicates.

Extraction Buffer 2A is a mild extraction reagent which can facilitate recovery of protein complexes. Thus,

protein yields obtained using Extraction Buffer 2A may be lower than those obtained using Extraction Buffer

2B (see Table 2 on p 9). Extracts can be concentrated with a Vivaspin 2 or Vivaspin 500 Ultrafiltration

Centrifugal Device.

Large volumes of TM-PEK 2B reagent can interfere with protein migration in SDS-PAGE. If high resolution

is desired, Extraction Buffer 2B samples for SDS-PAGE may be prepared in 2X SDS sample buffer.

Alternatively, employ a buffer exchange step.

For most transmembrane proteins, a 2-fold dilution of TM-PEK Reagent A or B in Extraction Buffer 2

results in efficient extraction. For some transmembrane proteins, extraction efficiency may be improved by

using TM-PEK Reagent A or B at a different concentration, from undiluted to a 10-fold dilution in

Extraction Buffer 2.

Protocol

1.Prepare Extraction Buffer(s) 2A and/or 2B by diluting the appropriate TM-PEK Reagent 2-fold with

Extraction Buffer 2. For membrane protein extraction from 1.0–5.0 x 107 cells, 0.2 ml of Extraction

Buffer 2A or 0.2 ml of Extraction Buffer 2B is required.

- To prepare 0.2 ml of Extraction Buffer 2A, mix 0.1 ml Extraction Buffer 2 and 0.1 ml

TM-PEK Reagent A.

- To prepare 0.2 ml of Extraction Buffer 2B, mix 0.1 ml Extraction Buffer 2 and 0.1 ml

TM-PEK Reagent B.

Notes: We recommend testing both Extraction Buffer 2A and Extraction Buffer 2B to determine which is optimal for your protein of interest.

Samples prepared with Extraction Buffer 2A may require concentration, depending on

downstream application.

High levels of Reagent B can interfere with protein migration on SDS-PAGE. Buffer exchange or

dilution in sample buffer may be required.

A 2-fold dilution of TM-PEK Reagent A or

B in Extraction Buffer 2 results in an efficient extraction

of most proteins, but reagent dilutions may be optimized (from undiluted to a 10-fold dilution).

1.Transfer 1.0–5.0 x 107 cells to a centrifuge tube.

2.Centrifuge cells at 1000 × g for 5 min at 4°C. (Alternatively, collect cells by centrifuging at

500 × g for 10 min at 4°C.)

3.Discard supernatant and gently resuspend cells in 5 ml PBS (4°C).

4.Centrifuge cells at 1000 × g for 5 min at 4°C.

5.Repeat Steps 3 and 4 two times, for a total of three washes.

6.Centrifuge cells at 1000 × g for 5 min at 4°C.

Note: At this point, cells may be frozen in liquid nitrogen and stored at –70°C.

7.Resuspend cells in 1 ml Extraction Buffer 1 + 5 μl Protease Inhibitor Cocktail Set III.

8.Incubate 10 min at 4°C with gentle agitation to avoid formation of cell clumps.

9.Centrifuge at 1000 × g for 5 min at 4°C.

10.Carefully remove supernatant. Store on ice. This is referred to as the ‘cytosolic (soluble)’ protein

fraction.

11.Resuspend pellet in 0.2 ml Extraction Buffer 2A + 5 μl Protease Inhibitor Cocktail Set III

or 0.2 ml Extraction Buffer 2B + 5 μl Protease Inhibitor Cocktail Set III.

12.Incubate for 45 min at room temperature with gentle agitation.

Note: The length and temperature of the incubation at Step 12 can be varied to improve extraction efficiency or to preserve target protein activity. Increasing the incubation time (up to 120 min) can

increase the protein recovery, but may result in decreased target protein activity. Conversely,

incubation at low temperature (4°C) may better preserve activity, but may lower the extraction

efficiency.

13.Centrifuge at 16,000 × g for 15 min at 4°C.

14.Transfer supernatant, which is enriched in integral membrane proteins, to a fresh tube.

Note: Store cytosolic and membrane fractions on ice if they will be analyzed on the same day. For long-term storage, dispense into aliquots and store at –20°C.

15.Determine the total protein concentration of the cytosolic and membrane protein fractions with the BCA

assay.

Note: For some cell types, total protein concentration of the membrane protein fraction (Step 15 above) will be >1.0 mg/ml. A two- to four-fold dilution in sterile, deionized water may be required to bring

the concentration of these samples within the linear region of the BCA standard curve.

Extraction of membrane proteins from tissue

Considerations Before You Begin

The following protocol is optimized for extracting membrane proteins from 25–50 mg of fresh or frozen

tissue. If tissue is not limiting, start with 100–1000 mg of tissue to offset sample loss during homogenization.

When using > 50 mg of tissue, increase volumes of the extraction buffers (see Table 1 on p 3 for buffer

volume guidelines). As an example, ~2 mg total protein can be extracted from 35 mg bovine liver (see Table

3 on p 9 in the Appendix for representative yields from other tissue sources and types). Yields from various

tissue types can vary considerably, however. Certain transmembrane proteins are expressed at very low

levels in various tissues, and thus may be below the lower limit of detection by immunological methods. In

this circumstance, analysis by mass spectrometry may prove beneficial.

Protocol

1.Prepare membrane Extraction Buffer(s) 2A and/or 2B by diluting the appropriate TM-PEK Reagent 2-

fold with Extraction Buffer 2. For membrane protein extraction from 25–50 mg of tissue, 0.2 ml of

Extraction Buffer 2A or 0.2 ml of Extraction Buffer 2B is required.

- To prepare 0.2 ml of Extraction Buffer 2A, mix 0.1 ml Extraction Buffer 2 and 0.1 ml

TM-PEK Reagent A.

- To prepare 0.2 ml of Extraction Buffer 2B, mix 0.1 ml Extraction Buffer 2 and 0.1 ml

TM-PEK Reagent B.

Note: We recommend testing both Extraction Buffer 2A and Extraction Buffer 2B to determine which is optimal for your protein of interest.

Samples prepared with Extraction Buffer 2A may require concentration, depending on

downstream application.

High levels of Reagent B can interfere with protein migration on SDS-PAGE. Buffer exchange or

dilution in sample buffer may be required.

A 2-fold dilution of TM-PEK Reagent A or

B in Extraction Buffer 2 results in an efficient extraction

of most proteins, but reagent dilutions may be optimized (from undiluted to a 10-fold dilution).

2.Ensure that all buffers are thawed and well mixed. Keep Extraction Buffers 1, 2A, and/or 2B on ice

during the extraction procedure. Keep the Protease Inhibitor Cocktail Set III at room temperature to

prevent DMSO from freezing.

3.Following dissection of the tissue of interest, quickly remove unwanted materials

(e.g., connective tissue, fat, blood vessels, etc). To slow proteolysis, keep tissue at 4°C while refining

the dissection.

4.Quickly slice the tissue into ~2 mm3 pieces. Add tissue slices to a tube containing 2 ml ice-cold PBS.

5.Gently flick tube to dislodge blood cells and other loosely attached material.

6.Collect tissue pieces by centrifuging at 100 × g for 2 min at 4°C. Remove and discard the supernatant.

7.Repeat Steps 5 and 6 for a total of two washes. After the second wash, ensure that all PBS has been

removed completely.

Notes: At this point, the tissue can be frozen on liquid nitrogen and stored at –70°C.

During tissue extraction, it is important to work quickly, but carefully. Keep the sample cool (<4°C)

and store buffers on ice throughout the extraction procedure.

8.Transfer the tissue (fresh or frozen) to a pre-cooled homogenizer. We recommend ideally, a glass

Potter-Elvehjem or Dounce homogenizer.

9.Add 5 μl Protease Inhibitor Cocktail Set III to the wall of the homogenizer.

10.Add 2 ml ice-cold Extraction Buffer 1 to the homogenizer.

11.Carefully homogenize until tissue is completely homogenized and intact pieces are no longer visible.

Use as few strokes as possible (e.g., ~10 strokes for 50 mg mouse liver). The number of strokes will

depend on the type of tissue used. If desired, homogenization efficiency can be monitored by phase

contrast microscopy. An efficient homogenization should generate small cell clumps rather that

fragmentation of individual cells.

Note: Some tissues (e.g., heart, muscle, brain) may be difficult to completely dissociate by mechanical homogenization. As an alternative, the ProteoExtract?Tissue Dissociation Buffer Kit

(Cat. No. 539720) including collagenase may be used. Other tissue-specific protocols may be

compatible with the TM-PEK kit.

12.Incubate 10 min at 4°C with gentle agitation.

13.Centrifuge at 1000 × g for 5 min at 4°C.

14.Carefully remove supernatant. Store on ice. This is referred to as the ‘cytosolic (soluble)’ protein

fraction.

15.Add 5 ml ice-cold PBS. Gently resuspend pellet. Collect membranes by centrifuging at 1000 × g for 5

min at 4°C. Carefully remove supernatant.

Note: When using > 200 mg tissue, perform an extra wash at this point to remove additional cytosolic proteins. Repeat Step 15 for a total of two washes.

https://www.wendangku.net/doc/6e16476890.html,pletely and carefully resuspend pellet in 0.2 ml Extraction Buffer 2A + 5 μl of Protease Inhibitor

Cocktail Set III

or 0.2 ml Extraction Buffer 2B + 5 μl Protease Inhibitor Cocktail Set III.

17.Incubate 15 min at 4°C with gentle agitation to avoid formation of cell clumps.

18.Centrifuge at 16,000 × g for 15 min at 4°C.

19.Transfer supernatant, which is enriched in integral membrane proteins, to a fresh tube.

Note: Store cytosolic and membrane fractions on ice if they will be analyzed on the same day.

For long-term storage, dispense into aliquots and store at –20°C.

20.Determine the total protein concentration of the cytosolic and membrane fractions with the BCA assay. Note: For some tissue types, total protein concentration of the membrane protein fraction (Step 19 above) will be >1.0 mg/ml. A two- to ten-fold dilution in sterile, deionized water may be required to

bring the concentration of these samples within the linear region of the BCA standard curve.

Frequently Asked Questions

Question Answer

How do I determine the protein

concentration of the membrane protein

extract? The components in the extraction buffers are directly compatible with common protein assays. We recommend using the BCA Protein Assay Kit (Cat. No. 71285-3). Note that the Extraction Buffer 2B fraction may require a 2-4-fold dilution to bring the total protein concentration within the linear

range of the BCA assay.

How do I prepare the TM-PEK fraction for one-dimensional SDS-PAGE? The TM-PEK fractions can be analyzed directly by one-dimensional SDS-PAGE. For samples

extracted with Reagent A, add SDS-PAGE sample buffer to a 1X final concentration and load

directly on to the gel. Samples extracted with Reagent B should be prepared using a 2X

concentration of SDS-PAGE sample buffer.

How can I concentrate the TM-PEK extracted proteins? It is possible to reduce the volume of Extraction Buffer 2A or 2B used, but this may decrease the

total protein yield. We recommend using the ProteExtract ? Protein Precipitation Kit (Cat. No.

539180) to concentrate samples for use in downstream applications not requiring native protein.

For applications that do require native protein, we recommend using an ultrafiltration device such

as Vivaspin 2 or Vivaspin 500.

How should the membrane fractions be treated prior to mass spectrometry analysis?

The membrane extracts should be applied to a one-dimensional or two-dimensional gel, spots or

bands cut from the gel, and then digested with trypsin.

Appendix Example extractions

In the examples below, the SDS extract (total cell lysate) serves as a positive control. The Extraction Buffer 2A recovers EGFR, which has a single transmembrane span, with efficiency comparable to TRITON ? X-100. However, Extraction Buffer 2A recovers Frizzled-4 and CELSR-3, both of which contain seven transmembrane domains, with far greater efficiency than does TRITON X-100.

A: Figure 1. Ex ction of transmembrane prote EGFR

B: Frizzled-4 C: CELSR-3

tra ins from MDA-MB-468 breast adenocarcinoma cultured ells. Transmembrane proteins were extracted from MDA-MB 468 cells using the TM-PEK kit. In the first two identical pools of 1 x 107 cells were treated with Extraction Buffer 1, which recovers proteins from the cytosolic fraction. The insoluble material was then treated with TM-PEK Extraction Buffer 2A or 0.5% TRITON ? X-100. Lane 1 shows the expected size of the target proteins and is not a quantitative control. Lanes 2–4 contain the extracted protein from 1 x 106 cells. Fractions were separated using a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were blocked and incubated with primary antibody to EGFR (panel A), Frizzled-4 (panel B) or CELSR-3 (panel C). Blots were developed using an HRP-conjugated secondary antibody and a chemiluminescent substrate. Lane 1, 0.5% SDS (total cell lysate); Lane 2, cytoplasmic fraction; Lane 3, membrane fraction (TRITON X-100); Lane 4, membrane fraction (Extraction Buffer 2A). Arrows indicate size of the full-length version of each protein.

c step,

Examples of total protein yields using TM-PEK

The values presented in Tables 2 and 3 below are intended to serve as a guide to estimate the required amount of starting material. For each cultured cell type, proteins were extracted according to the TM-PEK protocol from cell monolayers grown to 80% confluency in a T-75 flask. All tissues were disrupted mechanically using a Dounce homogenizer. Table 2. Protein yields for cytosolic and membrane fractions recovered from cultured cells using the TM-PEK kit.

Total Protein (mg/10 cells)*

Cell Type

Buffer 1

(cytosolic) Buffer 2A (membrane) Buffer 2B (membrane) Extraction

Extraction Extraction MDA-MB-468 (breast adenocarcinoma)

0.62 0.15 0.78 MCF 7 (breast adenocarcinoma)

0.68 0.26 1.27 A-431 (epidermoid carcinoma)

0.55 0.12 0.75 CHO-K1 (Chinese hamster ovary)

0.52 0.22 0.94 NCI-H292 (mucoepidermoid carcinoma)

0.06 0.04 0.47 HEP-G2 (hepatocellular carcinoma)

0.97 76 0.13 0.Mia PaCa-2 (pancreatic carcinoma)

0.23 0.05 0.52 HCT 116 (colon carcinoma) 0.60 0.10 0.76 *Protein concentrations were determined using the BCA .

Table 3. olic and membrane fractions recovered from mouse tissue Total Protein g/mg tissue)*

assay

Protein yields for cytos TM-PEK kit.

s using the (μTissue Type

Extraction

B

(cytos Extraction Bu A (me ne) Extraction Bu B (memb uffer 1olic)ffer 2mbra ffer 2rane)Liver 55.6 1.7 11.4 Heart

28.2 2.9 17.3 Brain

18.4 0.9 5.8 Spleen 31.6 8.2 8.8 §

Skeletal Muscle

13.6 2.1 6.3 Kidney 33.6.9 5.4 16*Protein concentrations were determined using the B §Average of two tr CA assay.

ials.

高纯度质粒小量快速提取试剂盒操作方法及步骤说明书

杭州昊鑫生物科技股份有限公司 htpp://https://www.wendangku.net/doc/6e16476890.html, HighPure Plasmid Mini Kit 高纯质粒小量快速提取试剂盒 目录号：PL03 试剂盒组成、储存、稳定性： 试剂盒组成保存 50次 （PL0301） 100次 （PL0302） 200次 （PL0303） 平衡液室温5ml 10ml 20ml RNaseA（10mg/ml）-20℃150μl 250μl 500μl 溶液P1 4℃15 ml 25 ml 50 ml 溶液P2 室温15 ml 25 ml 50 ml 溶液P3 室温20 ml 35 ml 70 ml 去蛋白液PE 室温16ml 31.5 ml 63 ml 第一次使用前按说明加指定量乙醇 漂洗液WB 室温15 ml 25ml 50ml 第一次使用前按说明加指定量乙醇 洗脱缓冲液EB 室温10ml 15ml 20ml 吸附柱AC 室温50个100个200个 收集管（2ml）室温50个100个200个 本试剂盒在室温储存12个月不影响使用效果。 储存事项: 1.第一次使用时，将试剂盒所带的全部RNase A加入溶液P1后（终浓度100ug/ml） 置于2-8℃保存。如果溶液P1中RNase A失活，提取的质粒可能会有微量RNA 残留，在溶液P1中补加RNase A即可。 2.环境温度低时溶液P2中SDS可能会析出浑浊或者沉淀，可在37℃水浴加热几分 钟，即可恢复澄清，不要剧烈摇晃，以免形成过量的泡沫。 3.避免试剂长时间暴露于空气中产生挥发、氧化、pH值变化，各溶液使用后应及时 盖紧盖子。 产品介绍：

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3.避免试剂长时间暴露于空气中产生挥发、氧化、pH值变化，各溶液使用后应及时 盖紧盖子。 产品介绍： 本试剂盒根据全血特点采用几个快速步骤提取基因组DNA。首先红细胞裂解液裂解去除不含DNA的红细胞，细胞核裂解液裂解白细胞释放出基因组DNA，然后蛋白沉淀液选择性沉淀去除蛋白，最后纯净的基因组DNA通过异丙醇沉淀并重溶解于DNA 溶解液。 产品特点： 1.从十几个配方中优选出的红细胞裂解液配方，裂解快速完全。 2.不需要使用有毒的苯酚等试剂。 3.快速，简捷，单个样品操作一般可在1小时内完成。 4.结果稳定，产量高（典型的产量10ml全血可提取出150-500μg），OD260/OD280 典型的比值达 1.7～1.9，长度可达50kb-150kb，可直接用于构建文库、PCR、Southern-blot和各种酶切反应。 注意事项 1.所有的离心步骤均在室温完成，使用转速可以达到2,500 x g，并配备容纳50ml心 管转头的传统台式离心机。 2.用户需自备异丙醇和70％乙醇。 3.典型的产量10ml全血可提取出150-500μg基因组DNA（不同样品尤其疾病样品中 中白细胞数量差异可能非常大，因此产量的个体差异也可能非常大）。 4.本试剂盒为溶液型，可以很容易的按照比例扩大或者缩小每次处理的全血量 （20μl-10ml），请联系我们索取其它处理量的操作手册。

小鼠cfos试剂盒使用方法

小鼠c-fos试剂盒使用方法 检测范围：96T 20pg/ml-480pg/ml 使用目的： 本试剂盒用于测定小鼠血清、血浆及相关液体样本中c-fos含量。 实验原理 本试剂盒应用双抗体夹心法测定标本中小鼠c-fos水平。用纯化的小鼠c-fos抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入c-fos，再与HRP标记的c-fos抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的c-fos呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算样品中小鼠c-fos 浓度。 试剂盒组成 1 30倍浓缩洗涤液20ml×1瓶7 终止液6ml×1瓶 2 酶标试剂6ml×1瓶8 标准品（960pg/ml）0.5ml×1瓶 3 酶标包被板12孔×8条9 标准品稀释液 1.5ml×1瓶 4 样品稀释液6ml×1瓶10 说明书1份 5 显色剂A液6ml×1瓶11 封板膜2张 6 显色剂B液6ml×1/瓶12 密封袋1个 标本要求 1．标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。若不能马上进行试验，可将标本放于-20℃保存，但应避免反复冻融 2．不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酶的（HRP）活性。 操作步骤 1.标准品的稀释：本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行稀 释。 480pg/ml 5号标准品150μl的原倍标准品加入150μl标准品稀释液 240pg/ml 4号标准品150μl的5号标准品加入150μl标准品稀释液 120pg/ml 3号标准品150μl的4号标准品加入150μl标准品稀释液 60pg/ml 2号标准品150μl的3号标准品加入150μl标准品稀释液 30pg/ml 1号标准品150μl的2号标准品加入150μl标准品稀释液 2.加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、标准孔、 待测样品孔。在酶标包被板上标准品准确加样50μl，待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样品最终稀释度为5倍）。加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。 3.温育：用封板膜封板后置37℃温育30分钟。 4.配液：将30倍浓缩洗涤液用蒸馏水30倍稀释后备用 5.洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此 重复5次，拍干。 6.加酶：每孔加入酶标试剂50μl，空白孔除外。 7.温育：操作同3。

高纯度质粒小提试剂盒使用说明书

高纯度质粒小提试剂盒 Pure Mini Plasmid Kit (目录号：HS0103) 产品包装 试剂盒成分 50 preps Buffer P1 15 ml Buffer P2 15 ml Buffer P3 20 ml Buffer PD 30 ml RNase A(10 mg/ml) 150 ul Buffer PW 60 ml Buffer EB 10 ml Spin Columns PA 50个 Collection Tubes (2 ml) 50个 （注意：使用前将全部RNase A 溶液加到Buffer P1中混合均匀，2 ~ 8℃保存） 保存条件 本试剂盒在室温(15 ~ 25℃)干燥条件下，可保存12个月；更长时间的保存可置于2 ~ 8℃。若溶液产生沉淀，应在使用前置于37℃下溶解沉淀。单独包装的RNase A 在室温可稳定保存12个月。加入RNase A 后的Buffer P1应置于2 ~ 8℃保存，可稳定保存6个月。 产品简介 本试剂盒用于高纯度质粒DNA 的小量制备与纯化。菌体经碱裂解、高盐、低pH 处理，质粒可从菌体中释放出来，并特异、高效地被离心柱硅胶膜（Spin Columns PA ）吸附。通过去蛋白液（Buffer PD ）和漂洗液（Buffer PW ）的清洗可去除蛋白及其他杂质，在低盐、高pH 条件下洗脱，最后得到高纯度的质粒DNA 。 北京厚生博泰科技有限公司 Beijing Hooseen Biotech Co., Ltd.

使用本试剂盒可从1 ~ 5 ml过夜培养的菌液中纯化得到高达20 ug的高纯度质粒DNA，可在30 min之内完成提取任务。所得质粒可直接用于酶切、转化、PCR、测序、低敏感细胞株的转染等各种常规分子生物学操作。 产品特点 1. 快速：步骤少，操作简便，节约时间。 2. 简便：离心吸附柱不需要预平衡，漂洗液Buffer PW 和去蛋白液Buffer PD不需要另加乙醇，即开即用。 3. 纯度高：所得质粒可直接用于酶切、转化、PCR、测序、低敏感细胞株的转染等各种常规分子生物学操作。 操作步骤 1. 取1 ~ 5 ml过夜培养的菌液，室温12,000 rpm离心1 min，尽量将上清去除干净。 （注意：根据菌液的浓度决定取液量，浓度高时取1 ml菌液离心即可，浓度低时可多收集一次） 2. 加入250 ul Buffer P1，用枪头充分吹打使菌体重悬均匀。 （注意：是否将RNase A溶液加到Buffer P1中并混合均匀；菌体沉淀是否悬浮充分，如有未彻底悬浮的菌块会影响裂解，导致提取的质粒浓度及纯度降低） 3. 加入250 ul Buffer P2，温和颠倒混匀6 ~ 8次，直到溶液变得清亮粘稠。 （注意：不可剧烈震荡，以免造成基因组DNA片段的污染，所用时间不要超过5 min，以免质粒受到破坏，如未完全变得清亮，可能是菌体太多，可增加Buffer P2的用量，在后续的操作中Buffer P3的用量也要相应增加） 4. 加入350 ul Buffer P3，立即温和颠倒混匀6 ~ 8次，可见白色沉淀物产生，室温静置2 min，然后12,000 rpm离心3 ~ 5 min。 （注意：Buffer P3加入后应立即混合，避免产生局部沉淀，如果上清中还有微小白色沉淀，可再次离心后取上清） 5. 小心将上清液转移到离心吸附柱Spin Columns PA中，静置2 min，让质粒DNA与吸附柱中的硅胶膜充分结合。12,000 rpm离心0.5 ~ 1 min，弃收集管中的废液。 6. 向吸附柱中加入500 ul去蛋白液，12,000 rpm离心1 min，弃收集管中废液。 （注意：如果宿主菌是endA-，如DH5α若TOP10，此步骤可省略。如果宿主菌是endA+，如TG1、BL21、HB101、JM101等，此步骤不可省略，因这些宿主菌含有大量的核酸酶，易降解质粒。如果提取低拷贝质粒

蛋白组分抽提试剂盒说明书

Overview ?Description ?References ?Product Information ?Applications ?Biological Information ?Safety Information ?Product Usage Statements ?Storage and Shipping Information ?Supplemental Information ?Prix & Disponibilité Prix & Disponibilité Référence DisponibilitéConditionnement QtéPrix Quantité 539790-1KIT En stock Contacter le Service Clients 1 kit à la validation de la commandePlus d'informations —Ajouter aux favoris Ajouter au panier Description

Overview Fast and reproducible extraction of subcellular proteomes from mammalian cells ProteoExtract? Subcellular Proteome Extraction Kit (S-PEK) is designed for fast and reproducible extraction of subcellular proteomes from adherent and suspension-grown mammalian cells. The S-PEK takes advantage of the different solubilities of certain subce compartments in the four selected reagents. In the case of adherent cells, the procedure is performed directly in the tissue culture dish without the need for cell removal. Cells or the parts of the cells remain attached to the plate during sequential extraction of subcellular compartments, until the appropriate extraction reagent is used. Thus, the early destruction the cellular structure by enzymatic or mechanical detachment of cells from the tissue cultu plate and any mixing of different subcellular compartments is prevented. For suspension-grown cells, extraction starts with gentle sedimentation and washing of the cel The stepwise extraction delivers four distinct protein fractions from one sample: ?Cytosolic fraction (F1) ?Membrane/organelle protein fraction (F2) ?Nucleic protein fraction (F3) ?Cytoskeletal fraction (F4) Proteins are obtained in the native state making the S-PEK suitable for many downstream applications such as 1D and 2D gel electrophoresis, immunoblotting, enzyme activity assa and protein microarrays. Sample size: 3-5x106or 25-50 mg tissue. Catalogue Number 539790 Brand Family Calbiochem? Synonyms S-PEK Kit Features and benefits ?Stepwise extraction resulting in four distinct subcellular proteomes from one sample ?Highly reproducible ?No ultracentrifugation steps ?Fast—needs just 2 hours with 45 minutes hands-on time ?Produces proteins suitable for functional studies

P0033 细胞膜蛋白与细胞浆蛋白提取试剂盒

细胞膜蛋白与细胞浆蛋白抽提试剂盒 产品简介： 碧云天的细胞膜蛋白与细胞浆蛋白抽提试剂盒(Membrane and Cytosol Protein Extraction Kit)提供了一种比较简单、方便地从培养细胞或组织中抽提细胞膜蛋白和细胞浆蛋白的方法。抽提的膜蛋白不仅包括质膜上的膜蛋白，也包括线粒体膜、内质网膜和高尔基体膜等上的膜蛋白。 本试剂盒通过匀浆适度破碎细胞，经低速离心去除细胞核和少数未破碎的细胞产生的沉淀，随后取上清高速离心获得细胞膜沉淀和含有细胞浆蛋白的上清，然后通过优化的膜蛋白抽提试剂从沉淀中抽提获取膜蛋白。 约90分钟即可完成培养细胞或组织的细胞膜蛋白与细胞浆蛋白的分离和抽提。抽提得到的蛋白可以用于SDS-PAGE，Western、酶活性测定等后续实验。 膜蛋白抽提试剂中含有蛋白酶抑制剂、磷酸酯酶抑制剂和EDTA等，后续不适合用于蛋白酶、磷酸酯酶等受这些抑制剂影响的酶的活性测定，但抽提获得的膜蛋白或细胞浆蛋白适合用于检测蛋白的磷酸化水平。 本试剂盒按照本说明书的操作步骤可以抽提100个细胞或组织样品。 保存条件： -20℃保存，一年有效。 注意事项： 需自备PMSF。PMSF一定要在抽提试剂加入到样品中前2-3分钟内加入，以免PMSF在水溶液中很快失效。 PMSF(ST506)可以向碧云天订购。 使用本试剂盒抽提到的细胞膜蛋白与细胞浆蛋白均可直接用碧云天生产的BCA法蛋白浓度测定试剂盒(P0009/P0010/P0010S/P0011/P0012/P0012S)测定蛋白浓度。抽提获得的细胞膜蛋白不适合用Bradford法测定蛋白浓度。 为了您的安全和健康，请穿实验服并戴一次性手套操作。 使用说明： 1.准备试剂：室温融解并混匀膜蛋白抽提试剂A和B，融解后立即置于冰浴上。取适量的膜蛋白抽提试剂A和B备用，在 使用前数分钟内加入PMSF，使PMSF的最终浓度为1mM。 2.准备细胞或组织样品： a. 对于细胞 (1) 收集细胞 对于贴壁细胞：培养约2000-5000万细胞，用PBS洗一遍，用细胞刮子刮下细胞或用含有EDTA但不含胰酶的细胞消化液处理细胞使细胞不再贴壁很紧，并用移液器吹打下细胞。离心收集细胞，吸除上清，留下细胞沉淀备用。尽量避免用胰酶消化细胞，以免胰酶降解需抽提的目的膜蛋白。 对于悬浮细胞：培养约2000-5000万细胞，直接离心收集细胞，吸除上清，留下细胞沉淀备用。 (2) 洗涤细胞：用适量冰浴预冷的PBS轻轻重悬细胞沉淀，取少量细胞用于计数，剩余细胞4℃，600g离心5分钟沉淀 细胞。弃上清，随后4℃，600g离心1分钟，以沉淀离心管管壁上的残留液体并进一步沉淀细胞，尽最大努力吸尽残留液体。 (3) 细胞预处理：把1毫升临用前添加了PMSF的膜蛋白抽提试剂A加入至2000-5000万细胞中，轻轻并充分悬浮细胞， 冰浴放置10-15分钟。 b. 对于组织： 取约100毫克组织，用剪刀尽量小心剪切成细小的组织碎片。加入1毫升临用前添加了PMSF的膜蛋白抽提试剂A，轻轻悬浮组织碎片，冰浴放置10-15分钟。注：如果组织样品比较少，也可以使用更少的组织量，例如30-50mg，后续试剂的用量及操作步骤不变；组织用量较少时，最后获得的膜蛋白也较少。

质粒提取试剂盒 说明书 翻译

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