Hi Everyone,
I am a beginner at eukaryotic(HEK293T) protein expression. I am trying to purify a HIS tagged 200 kDa viral polyprotein using pCDNA3.1 vector. I am not sure which lysis buffer should I use? and Should I sonicate to lyse cells or not?
Hi Abhijeet Singh,
I use a simple modification of RIPA buffer(having multiple detergents) for a broad range of cell lines including HEK293T and seems to work really well. I too am looking for a high-molecular weight protein of 270kDa and RIPA works well for the purpose.
Akhil Baby Hi Akhil,
Thank for your reply. Are you trying to purify the 270 kDa protein of interest ?
I have one more query, what percentage of SDS PAGE gels do you use to run 270 kDa samples on gel ?
Also, after lysing cell with RIPA buffer, do you still do sonication for lysing cells further ?
Thank you so much !!
Abhijeet Singh
I just pass the lysate through a 22G needle syringe, no sonication is required. Actually, my work doesn't require purification of the 270kDa protein. I usually use a 4-20% gradient Tris-Gel since I need separation of the phosphorylated and non-phosphorylated forms of the protein and also other proteins of different molecular weight. But if you do not have such requirements you can use a 10 or 12% gel.
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