I am trying to do 2D gel on mammalian cell protein. I extract the protein by ripa buffer and then do tca-acetone precipitation and then load the protein (200ug) onto the gel and continue with manufacture's protocol (invitrogen). But still I am getting gels like the attached photograph.
Hi Faruk,
Your samples preparation must be optimized. Work with fresh starting material tissues for proteins extraction. Avoid 6 mouths stored tissues at -80C. there is always degradation of proteins even at freeze temperatures.
After tca-ac precipitation, do not over dry your pellet. Also do not heat your prep when re-suspending it. Use 8M Urea for resuspension in the resupension buffer.
Start with these tips first; it improve considerably the quality of the gels.
Good luck
Your first dimension is not well focused. What is the pH rang you are using?
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