I am now working on the Co-immunoprecapitation of human sperm. I have tried some lysis buffers such as RAPI, lysis buffer purchased from Thermo and standard IP lysis (50mM tris, 150mM NaCl, PH 7.4). However, the lysate concentrations were very low. Is there the protocol or usable lysis buffer in human sperm? Thank you for your kind suggestion.
If what you mean is that protein concentration is too low, you can try to gently concentrate your sample with one of the many methods available (spin columns, etc).
If you can elaborate on what you need to do with the samples you obtain, we might be able to provide a more articulate answer.
Thank you for your suggestions, Carlo. Here is steps when I lysated the samples:
1.Human sperm was isolated by gradient centrifugation.
2. Wash sperms (6 patients, around 10^9 sperm cells/patients ) once in PBS, add 3ml ice-cold IP Lysis/Wash Buffer with 2x proteinase inhibitors.
3.Incubate mixer for 2 hrs at 4 ˚C with head-over-head rotation. Remove cell debris by centrifugation at ~13,000 × g for 10 minutes.
4. The supernatant was measured the protein concentration, but we only can get less 500ug/ml of the 3ml lysate (In our experiences in mammalian cells, the protein concentration should be about 6mg/ml / 3ml lysate).
Hi, Hao
I think with 500ug of protein (independently of the volume) you would be able to perform an IP. I use 200-400ug for IP with good results.
Your lysis buffer is not the problem, sperms always give low protein concentration.
Why do you use so many lysis buffer and incubate for so long at 4ºC (2 hours)?
Maybe you should try to perform the IP anyway.
Which protein do you want to observe? It is a low-expressed protein?