lysis buffer to extract transmembrane protein - (Jun/22/2006 )

Hi,

when I use lysis buffer to extract protein, I found that the peak is around A260 by spectrophotometry. I think A260 stands for the nucleic acid. I don't know this sample can be used for western blot. Does anybody have the same problem or have any idea to explain it?

Thank you very much!

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you just have one peak at 260?

what extraction buffer did you use?

if you ALSO have a peak at 260 it is presumably DNA contamination and you can try a DNAse digest (check if your buffer is compatible with enzyme)

you extracted from tissue or cell culture?

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hmm, don't know what went wrong:

I disrupt my tissue mechanically and by sonification in the lysis buffer
(there are varius types, but a biochemist once recommended especially for transmembrane proteins just 20 to 50 mM Hepes ph 7,4 plus 5 mM MgCl2)
and then spin down the debris at least two times always transferring the supernatant into new reaction tube.
I centrifuge at 4° C 30min 14.000g

Do you still have the pellet(s)?
Try to resuspend in lysis buffer and check if protein is in there...

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