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total cell protein vs membrane protein - (Sep/26/2010 )

I used MPER(pierce) for total cell proteins and RIPA buffer for membrane protein extractions. Total cell proteins and membrane proteins differ in their ELISA readings and Western blot band pattern even for the same specific protein from the same amount of starting sample. Why is that so? Is it because of the use of different buffers?

-srm mrs-

Hi srm mrs,
First of all if I am not wrong the RIPA buffer contains Sodium Deoxycholate and Nonidet p40 as its core ingredient, whereas the MPER buffer is a mild detergent base protein buffer. The reason you may be getting differed readings and bands may be because both Sodium Deoxycholate and Nonidet p40 perform a harsh treatment specifically on the cell surface proteins and cause a conformational change in the polarity of the cell surface proteins so that they can leave the membrane and get released in the liquid suspension. Thus the degree of polarity is different in the same specific protein from same amount of the same sample. Simply explaining would be the mildly extracted protein may be of secondary structure and the harshly extracted protein may be tertiary.

-dr junwen wang-

Hello dr junwen wang,
Thank you so much for quick reply. And the answer is also so simplified, are you from the same field? The reply tells me why there is a difference in the readings but does this mean that one of my readings is wrong? And if it is then how can know which one is wrong?

-srm mrs-

Hi srm mrs,
Donít mention it. I did not say that any of your reading is wrong. They both are correct. But if your research is basically based on comparing them then sorry to say your research itself may be wrong. But so long as you carry out the whole research with either one of them then it doesnít matter which one you choose. Generally I would choose the harshly treated membrane protein since there will be more chances of sensitivity even at smaller amounts. But the mildly extracted one is also good enough since you u get a spectrum of all proteins.

-dr junwen wang-

Hello dr junwen wang,
Thank you so much. I am not comparing them as my research; I have used the mild one for my research. With the membrane protein I was just verifying whether the ELISA is working out specifically. But thanks again, it was of great help.

-srm mrs-