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    plasma-membrane-protein-extraction-kit-ab65400.pdf

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Plasma Membrane Protein Extraction Kit (ab65400)

  • Datasheet
  • SDS
  • Protocol Booklet
Submit a review Q&A (88)References (110)

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Functional Studies - Plasma Membrane Protein Extraction Kit (ab65400)
  • Membrane protein assay - ab65400

Key features and details

  • Assay time: 1 hr

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Overview

  • Product name

    Plasma Membrane Protein Extraction Kit
  • Assay time

    1h 00m
  • Product overview

    Plasma Membrane Protein Extraction Kit ab65400 provides optimized buffers and reagents for effective extraction of plasma membrane proteins from mammalian tissues and cells.


    Other commercially available methods can only extract the total cellular membrane proteins (combinations of plasma and organelle membrane proteins). The ab65400 kit offers two options: either extract the total cellular membrane proteins, or purify the plasma membrane proteins specifically.


    The procedure offers consistent yield and high purity (over 90%). Membrane proteins prepared using the kit can be utilized in a variety of applications, such as Western blotting, 2-D gels, and enzyme analyses, etc. The entire procedure takes less than 1 hour.

  • Notes

    This product is manufactured by BioVision, an Abcam company and was previously called K268 Membrane Protein Extraction Kit. K268-50 is the same size as the 50 test size of ab65400.

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components Identifier 50 tests
    Homogenize Buffer NM 1 x 100ml
    Lower Phase Solution WM 1 x 20ml
    Protease Inhibitor Cocktail (Lyophilised) Red 1 vial
    Upper Phase Solution NM 1 x 20ml
  • Research areas

    • Kits/ Lysates/ Other
    • Kits
    • Molecular Biology Kits
    • Cell Fractionation Kits
    • Kits/ Lysates/ Other
    • Kits
    • Extraction Kit
    • Membrane
  • Relevance

    Membrane Protein Extraction is a method for extraction of membrane proteins from mammalian tissues and cells. Many procedures extract total cellular membrane proteins which is a combination of plasma and organelle membrane proteins.

Images

  • Functional Studies - Plasma Membrane Protein Extraction Kit (ab65400)
    Functional Studies - Plasma Membrane Protein Extraction Kit (ab65400)Kweon, Hae-Jin et al., Scientific reports?vol. 6 30684., Fig 1, doi:10.1038/srep30684
    Western blotting on the plasma membrane (PM) fraction extracted useing ab65400, total cellular membrane (TCM) fraction, and total lysate of cells expressing GFP-tagged ASIC2a or ASIC2b was performed using anti-GFP antibody. As controls, the PM and the TCM fractions were blotted using anti-calnexin (CNX) antibody, and total lysate was blotted using anti-GAPDH antibody.
  • Membrane protein assay - ab65400
    Membrane protein assay - ab65400Image from Choveau F.S et al., PLoS One 10(12), Fig 2C. Doi: 10.1371/journal.pone.0145367. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Cell membrane protein was collected using plasma membrane protein extraction kit (ab65400). The upper panels show the plasma membrane myc-tagged protein; the middle panels show the cytosolic myc-tagged proteins and the lower panels show the total myc-tagged protein from CHO (Chinese Hamster ovary) whole-cell lysates.

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (110)

Publishing research using ab65400? Please let us know so that we can cite the reference in this datasheet.

ab65400 has been referenced in 110 publications.

  • Chen Z  et al. Cell surface GRP78 regulates BACE2 via lysosome-dependent manner to maintain mesenchymal phenotype of glioma stem cells. J Exp Clin Cancer Res 40:20 (2021). PubMed: 33413577
  • Lin CN  et al. An aptamer interacting with heat shock protein 70 shows therapeutic effects and prognostic ability in serous ovarian cancer. Mol Ther Nucleic Acids 23:757-768 (2021). PubMed: 33614227
  • Miyata T  et al. Differential role of MLKL in alcohol-associated and non-alcohol-associated fatty liver diseases in mice and humans. JCI Insight 6:N/A (2021). PubMed: 33616081
  • Sadhukhan T  et al. In a mouse model of INCL reduced S-palmitoylation of cytosolic thioesterase APT1 contributes to microglia proliferation and neuroinflammation. J Inherit Metab Dis 44:1051-1069 (2021). PubMed: 33739454
  • Bhandari P  et al. GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals. Elife 10:N/A (2021). PubMed: 33913808
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 88 Abreviews or Q&A

Question

I just wanted to give an update on the replacement kit. I have had much much better success with recovering the plasma membrane fraction after we spoke on the phone. I still do not get 100% purity, but the contamination is not too bad. I never have contamination from the nuclear fraction (probing for p84), but occasionally small contamination when I probe with a cytosolic marker. I would recommend against using a housekeeping gene like tubulin as a cytosolic marker, as I always see that in my plasma membrane fraction, but the sample looks much cleaner when I use a different cytosolic marker (eg GSTpi). Finally, I think what helped the most in recovery was modifying the protocol. I would recommend to remove step 2 in Part B of the protocol, and any further steps (steps 5 and 6 in part B) that follow up from this step. I think when you try to get upper and lower phase from the mixed solution, its never as clean as it would be if you took it from the primary container, and this can lead to the sample ending up in the wrong phase because the volumes of the solution aren't as exact, especially because of viscosity differences in the upper/lower phase solutions. Also for researchers using tissue instead of cells as I am, the homogenization step is extremely important. I always use a tissue grinder before putting the sample in the dounce homogenizer, otherwise the debris pellet will be quite large.

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Abcam community

Verified customer

Asked on Mar 04 2016

Answer

Thank you for the update. I am glad to hear the replacement kit worked out better for you. Eliminating those extra steps will probably, as you found, give purer fractions but possibly at the cost of lower yield.

I am not sure why tubulin was getting pulled down with the membrane fraction; maybe it was complexed with membrane-bound proteins as suggested in the article at this page.

http://www.sciencedirect.com/science/article/pii/S0005273609001035

Biochim Biophys Acta. 2009 Jul;1788(7):1415-33.

Read More

Tom Ruyle

Abcam Scientific Support

Answered on Mar 04 2016

Question

confusion about the protocol. why does the upper reagent need to be taken out the bottle before adding into the tube again (step 3 to 5 in the protocol)

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Abcam community

Verified customer

Asked on Jun 28 2012

Answer

Merci pour votre requête. J'ai contacté le laboratoire et ils m'ont confirmé que cette étape est pour équilibrer la "upper phase" solution. Il ne faux donc pas prendre la solution directement de la bouteille.

Voici encore la description étape par étape du laboratoire:

a. you have your total membrane protein in both the phase solutions.

b. you are making an equal amount upper and lower phase solutions and normalizing/equilibrating each with the other.

c.you collect the upper phase solution containing the isolated memb protns.

d.you reisolate the pure memb protns, with equilibrated upper phase solution. If you take this solution from the stock it will not be equilibrated and might lead to lower efficiency if isolation.

J'espère que cette information clarifie le protocole. Veuillez ne pas hésiter à nous recontacter si vous avez des autres doutes ou questions.

Read More

Abcam Scientific Support

Answered on Jun 28 2012

Question

Can this kit be used to purify just the plasma membrane, rather than the plasma membrane proteins?

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Abcam community

Verified customer

Asked on Feb 15 2012

Answer

This kit may be useful for extracting and retainingthe plasma membrane fraction without the plasma membrane proteins but it has not been specifically designed for this purpose. Please let us know how things go if you do choose to use ab65400 for this experiment.

I hope this is helpful. Please contact us again if you have any further questions.

Read More

Abcam Scientific Support

Answered on Feb 15 2012

Question


1.       I would like to isolate a 4 transmembrane protein (504 amino acid) from plasma membrane using ab65400 Plasma Membrane Protein Extraction Kit (attached please find manual).


2.       It is very important for us to understand the basic idea how this isolation plasma membrane proteins kit work (for example: labeling PM with Biotin and isolation with SA or using sucrose etc.)


I appreciate if you could provide me this information.


In addition, do you have any experience with isolation a 4 transmembrane protein using this kit?

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Abcam community

Verified customer

Asked on Aug 31 2016

Answer

I can confirm that the kit uses an aqueous polymer two-phase system to separate plasma membranes. Most specifically it uses the PEG/ Dextran system and the plasma membrane tend to concentrate in the upper phase.


I am sorry we do not have specific data for isolation of 4 transmembrane proteins using this kit.  I would like to reassure you that the kit is covered by the Abpromise guarantee should it not work as expected.

Read More

Abcam Scientific Support

Answered on Aug 31 2016

Question

1. Can this kit be used with smaller cell numbers, such as 105 or 106? If so, what protocol changes are required, if any?
2. Is there a limit to the number of transmembrane domains a protein can have and still be solubilized with this kit?

Read More

Abcam community

Verified customer

Asked on Nov 19 2014

Answer

1. The smaller cell number can decrease yield. Hence it is at the user's discretion to reduce cell numbers. For some cells, based on size and density of expression of membrane proteins, smaller cell number can still yield enough membrane proteins. If cell number is reduced, reagents should be scaled down proportionally.
2. Proteins with domains spanning the plasma membrane multiple times can also be isolated by this kit. We have not specifically tested the effects specific number of TM domains domains have on membrane protein solubility using this kit.

Read More

Jeremy Kasanov

Abcam Scientific Support

Answered on Nov 19 2014

Question


1. I would like to isolate cell membrane protein and membrane associated protein but not mitochondrial membrane protein from fibroblasts.
2. Also It is important to keep protein with native form. Does the protease inhibitor cocktail also works for PADI? ( a enzyme catalyze the posttranslational modification reaction generating citrulline residues)
3. After isolation the membrane protein would be use for WB and IP.
Does this kit fits my requirements ?

Read More

Abcam community

Verified customer

Asked on Aug 07 2014

Answer



1. This kit isolates the plasma membrane associated proteins as well as transmembrane proteins on the plasma membrane. Organelle membrane proteins are not isolated. If total membrane proteins were desired, you would get all membrane proteins including those from mitochondria at step A 6.

2. The membrane proteins are preserved in their native state since there are no denaturing or harsh chemicals used in this kit. I am sorry we have not specifically looked for the peptidylarginine deiminases you are interested in.

3. The proteins isolated with this kit can be used in WB and IP.
.

Read More

Sam Washer

Abcam Scientific Support

Answered on Aug 07 2014

Question

I was looking at the plasma membrane protein extraction kit. I'm looking to analysis protein expression of a gene of interest on the surface of the membrane vs internalized gene. My question would be if this kit, would give me the sufficient results to obtain the answer to this possible question. Thanks

Read More

Abcam community

Verified customer

Asked on Jul 24 2014

Answer

Yes, this will give you two sub-cellular fractions: plasma membrane and cytosolic. So you will be able to demonstrate the relative amounts of your protein of interest in each fraction using an assay such as western blotting, assuming you have an antibody that can detect it.

Read More

Tom Ruyle

Abcam Scientific Support

Answered on Jul 24 2014

Question

Do you know if this kit extract intact-native proteins?

Read More

Abcam community

Verified customer

Asked on Feb 07 2014

Answer

The proteins isolated when using this kit should be in their native conformation.

Read More

Caitlin Valued Customer

Abcam Scientific Support

Answered on Feb 07 2014

Question

The homogenization step is defective, I have homogenized cells in Dounce homogenizer for 50 times, when I did not get a nice result, I put the sample in liquid nitrogen, then tried to grind the sample with the Dounce homogenizer pistol. Do you have any suggestion to improve the procedure?

Read More

Abcam community

Verified customer

Asked on Feb 07 2014

Answer

The frozen tissue cannot be pulverized with the Dounce homogenizer. This has to be done with a pulverizer or tissue grinder. Once ground, the buffer can be used to homogenize the bits in a Dounce homogenizer.

Fresh tissue can be homogenized directly and can require more passes than 50 sometimes depending on what tissue is used (muscles, cartilage etc take more passes).

Read More

Elisa Thomas

Abcam Scientific Support

Answered on Feb 07 2014

Question

I have tried the plasma membrane extraction kit again with no success! I still did not get any pellet at the end, and here are the details:

I am using T47D breast cancer cells.

I have used around 6x 108 cells; wet weight was around 1.05 gram, so this should be enough.

For homogenization I shocked freeze the sample in liquid nitrogen, and around 80% were lacking this shiny ring.

After centrifugation in step 4 of extraction of total cellular membrane proteins, I got a huge nuclear pellet, I collected the supernatant.

After centrifugation in step 5, I got a tiny brownish pellet; it was very small, but detectable.

Then I proceeded with steps as in the protocol.

At step 4 of purification of plasma membrane protein, I had a nice separation with turbid interface.

At step 7, I collected 300 µl of the combined upper phase, so I added 1500 µl distilled water and centrifuged, but then I got no pellet at all.

I even contacted one of the references (Ishikawa S et al. Role of connexin-43 in protective PI3K-Akt-GSK-3ß signaling in cardiomyocytes. Am J Physiol Heart Circ Physiol 302:H2536-44 (2012) ), and he kindly confirmed my steps, and stated that it is very straight forward with no tricks.

Read More

Abcam community

Verified customer

Asked on Feb 05 2014

Answer


You are obtaining a huge pellet after step 4. This is the low speed centrifugation at 700g and pellets down unbroken cells, nuclei and debris. Getting a huge pellet at this step is indicative of incomplete homogenization. The steps are correct but homogenization is key to getting good yields for membrane protein preps. No shiny nuclei can often be mistaken under the microscope for small cells.

Also, it is important to use the correct RPM values for the spins based on the g-forces mentioned on the datasheet. The pellet after step 7 can be small and translucent sometimes stuck to the tube. Once the 1500ul water is added, it is important to have enough space in the tube for centrifugation with the proper effects. If there is not enough space, the pellet might not be obtained. I would suggest separating the sample into 2 tubes with 900ul in each tube (300ul+1500ul water =1800ul total split into 2) and then spinning. This might yield better results.

Read More

Elisa Thomas

Abcam Scientific Support

Answered on Feb 05 2014

1-10 of 88 Abreviews or Q&A

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