Protein extraction

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Jaycee
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Protein extraction

Hello

I need help with protein extraction. I have extracted a soluble cytoplasmic proteins using Tris-HCl lysis buffer (20 mM Tris-HCl, pH 7.5 and 1 mM EDTA) and what i'm interested in is membrane bound proteins,so from here how will i go about this in order to get the protein of interest(membrane bound proteins)?

Jaycee

g a
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 Hi Jaycee

 Hi Jaycee
Will you analyze both membrane bound and cytosolic fraction as a whole in a single experiment? If yes then you can use Urea + Thiourea for  extraction and add a detergent which is non ionic or zwitter ionic along with a buffer - salt system.

In case you need to analyze both fractions separately, first lyse the cells and centrifuge at about 15000g to sediment membrane fraction and remove the soluble fraction. Follow this up with extraction of membrane proteins using a buffer-salt and detergent system.

You can check protocols specific to your cells and experimental needs.