Plasma膜蛋白Extraction试剂盒(ab65400)

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.

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.

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.

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.

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.

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.
.

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.

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

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).

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.