Lysing hela cells - poor protein recovery (May/29/2006 )
Hi
You might have some protein degradation BUT the main issue here is the inefficient lysis. From 5 T75 flasks you could get much more. I don´t know by heart but 2 mg is not a lot.
So I would like to know what is the percentage of NP40.
You said you add lysis buffer and vortex.
For how long do you incubate cells with lysis buffer?
You cannot simply vortex immediatly unless you sonicate your samples.
You can use different amount of detergent ranging from 0.1% to 1% but the lower the amount the longer should be the incubation ( always at 4C, preferably rotating).
Sonicate or use DNAase.
You said you add lysis buffer and vortex.
For how long do you incubate cells with lysis buffer?
You cannot simply vortex immediatly unless you sonicate your samples.
I use 1%. I do vortex immediately after adding lysis buffer.
Alright, I've redone the lysis and got about the same results.
This time I used a detachment buffer: Tris, NaCl, EDTA, 10mM B-merc at pH 7.0. The protocol: wash cells with this buffer twice, then leave in the buffer for 10-20min, collect, spin, resuspend in lysis buffer (Tris, NaCl, 1%NP40, 1mM PMSF, 10ug/ml Leupeptin, 10ug/ml Aprotinin). I left the resuspension on ice in the lysis buffer for 5min, vortexed, then spun at 13000g for 15 min at 4degrees to collect membranes, etc.
I used 7 completely confluent 175cm^2 flasks and got about 8mg. I guess Hela's don't give a lot of protein.
Dear Jaknight,
The protein levels you are getting are far far too low. In my experience the best 3 methods are Sonication, Freeze-thaw and Homogenistion. Sonication by probe will give you the best results. I have prepared induced enzymes from cell lines using this method for 15 years. The enzymes are fully functional and are used for drug screening programmes, so are not denatured. Here goes :-
Get cells on ice in an appropraite lysis buffer containing a protease inhibitor cocktail ( small volume)
Sonicate the cells for 5 seconds at an appropriate amptitude that you will need to optimise ( cell specific). Leave the cells to rest for 25 seconds, this allows the sonic probe to cool down and will prevent denaturation of protein.
Sonicate again for 5 seconds and rest for 25 seconds as above
Repeat once more.
This protocol gives me full lysis, look under the microscope to see cell debris. The enzymes released are fully functional when cofactors are addded back in the assay. You will however need to optimise for your cells i.e one cycle of sonication maybe enough, or you may need 5 cycles.
Hope this is useful
Rhombus (30 years in the Lab).
Thanks Rhombus.
It's not crucial that I get all the protein. What I have gotten is more than enough. I was just wondering why I get so little protein from the lysis.
It's not crucial that I get all the protein. What I have gotten is more than enough. I was just wondering why I get so little protein from the lysis.
May be the problem is with the lysis buffer. I used promega passive lysis buffer to lyse Hela cells and it worked well for me. Where as I had problems with NP40/Ripa buffers. I tried all the possible methods, like freeze thaw and sonication but they never gave good yield of proteins. With promega pssive lysis, i directly lyse the cells in cold conditions and scrape them and spin immediately and I found the yields are quite good.
rhombus - you mention freeze thaw - more info? does it work as well as sonicating? and what about for tissue? cheers....
Nah, try sonicating as some have suggested earlier. That's what I've done and it increased my yield skyrocketing.