I am having strange results with my Western blot results. My protein of interest is suppose to increase after treatment. Infact it has decreased compared to the control. Does this mean the proteins have went into insoluble fraction or there is something else happening? Some studies have shown that my protein of interest sometimes do go to insoluble fraction. Can someone help me explain this? Also if you have a detailed protocol to isolate insoluble fraction of protein, that would be a great help. Thank you.
I usually follow this procedure
http://wolfson.huji.ac.il/purification/Protocols/InclBodCont.html
I usually disrupt the induced and not-induced cell and centrifuge it. Then load both of fraction (supernatant and percipitate) on SDS_PAGE gel and compare the considered protein expression profiles with each other.
After protein induction and sonication. Centrifuge your bacterial lysate then collect the supernatant and the pellet. Resuspend your pellet with TBS and load the supernatant and the resuspended pellet in wells for SDS_PAGE and western blot analysis.
Thank you. So no need to perform protein quantification or add lysis buffer again to the pellets?
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