Prathibha, are you able to trace the extraction by Western Blot? I.e have each fraction from the extraction process on the same gel and do you see if your protein gets extracted or is it in the corresponding precipitate?

Hello Prathibha

I thing it is better to extract first the peripheral protein in stage one by using either different ionic strength or different pH then eluate the integral one with the buffer that contain detergent. Mind you that using SDS in the buffer will cause denaturation of the protein so the antibody may not precipitate your your protein in this case & to have an initial idea about the extracted proteins you must run a native electrophoresis then the SDS one .

You may read the Methods of Enzymology Volume 22 which deals with extraction of different membranes' proteins from different types of cells will help you in this subject.

Good luck.

It is better to narrow down the subject by investigating the problem. Is that related to extraction efficacy or immunoprecipitation and or visualization failure? Can you identify the proteins by blotting after extraction as Mr. Schechinger indicated?

Hello Wolfgang, Hathama and Ismail

Thank you for the information. I ran westerns from each fraction from the extraction process - here is the description of what I did

After the lysis, I treated my soluble fraction and membrane fraction with Anti-flag beads and did an overnight incubation.

Next day, before I began washing the beads, I took out 30 ul as pre-wash fraction. This is right after the IP.

Then washed my samples with lysis buffer once and with wash buffer 4 times. Took 20 ul from each wash.

Finally eluted using SDS-page loading buffer, cooked it for 3 minutes and ran on 8% SDS gel and western

From my westerns I dont see signal in my pre-wash and washes so I assume protein binding on the beads.

Please let me know if you think am going wrong in any of these steps.

Hathama Razooki Hasan - I will try running a Native PAGE in the coming days to check the extracted proteins