Hi. I tried both Glutaraldehyde and PFA crosslinking methods....

YY
Y
Jul 23, 2021
protocol Protocol: Biochemical Pulldown of mRNAs and Long Noncoding RNAs from Cellular Lysates Coupled with Mass Spectrometry to Identify Protein Binding Partners
Hi. I tried both Glutaraldehyde and PFA crosslinking methods. However, I found fixed cells are very difficult to lysis. How should determine the cells have been lysed well? best, Yang
answer Answer
recommend recommend recommend recommend Recommend
follow follow follow follow Follow
share share Share
1 answer
Sort by: most helpful / most recent

Anca Flavia Savulescu Author Answered Jul 26, 2021

University of Cape Town

Dear Yang,

Thank you for your inquiry.

We have not had issues with lysis with the specific cells and Lysis buffer that we tested this on. We have tried other Lysis buffers though, and they have worked to certain extents (some better than others).

What cell line or primary cells do you use? I think that it is worth trying in parallel a few lysis buffers, as some work better for some cells than others.

Also, I wanted to ask - what do you mean by difficult to lyse them - so there is no clear difference between the pellet and the supernatant? Is the protein concentration in the supernatant very low?

Hope this is helpful and please let me know if I can do anything else to help.

Anca
0 helpful
0 unhelpful
comment 0 comments down up
share Share

My answer

Write your answer...

References (optional)

add Add more

post Post an Answer