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Solubilizing conditions for brain lysate

ER protein EndoH PNGase

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3 replies to this topic

#1 Coomb Raider

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Posted 07 January 2013 - 04:17 AM

Dear Bioforums,
I have been making a cerebellar lysate from adult mouse using the following buffer:

3mM NaHEPES (pH 7.5), 320mM Sucrose, 0.1% Triton, PMSF, protease inhibitor tablets. We are finding the low salt concentration important for maintaining protein solubility.

I have some surprising results which I think can possibly be explained by a lack of ER proteins in our lysate. Our protein has no EndoH sensitivity and is not present in a non-glycosylated form, most unexpected. Similarly, we are getting very low yields of a protein we expect to be completely retained in the ER.

Does anybody have any advice about these buffer conditions and how to best solubilize both ER and plasma membrane proteins in low salt, or of course any alternative explanations.

Thanks very much
CR

#2 Curtis

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Posted 08 January 2013 - 08:15 PM

How do you clear the lysate? what is your protocol? how do you not find ER protein in your lysate? western?

#3 Coomb Raider

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Posted 09 January 2013 - 04:23 AM

Thanks for the reply Curtis!

We are currently trying a completely different protocol eliminating detergent from the initial buffer and following centrifugation using a harsher detergent to solubilize the whole pellet. I'm hoping this will fix the issues we've been having. I'll report back if we continue having problems

Thanks again
CR

#4 Curtis

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Posted 09 January 2013 - 08:33 PM

so you must be using hypotonic buffers, and looking for membrane proteins?




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