6 replies to this topic

[donny]

I'm trying to lyse small volumes of E coli cells, bind the proteins to IMAC resin, then elute them for SDS-PAGE analysis. I'm thinking of using a detergent-based lysis buffer like B-Per and BugBuster. I've tried 1% Triton-X in PBS but it doesn't seem to be lysing very well. I've used BugBuster before and I could see the cells clarify almost immediately and they use non-ionic detergent. Does anyone have any suggestion which detergents I should try and at what concentration? SDS is out as it's incompatible with IMAC resin.

[pDNA]

i would use Triton X-100 (0.1%) and lysozyme (0.2 mg/ml) in column buffer (i think something like 300 mM NaCl in 50 mM NaHPO4, pH 8) ...lysozyme will do the job pretty well and will not interfere with the IMAC.

Regards,
p

donny, on 12 February 2011 - 04:05 AM, said:

I'm trying to lyse small volumes of E coli cells, bind the proteins to IMAC resin, then elute them for SDS-PAGE analysis. I'm thinking of using a detergent-based lysis buffer like B-Per and BugBuster. I've tried 1% Triton-X in PBS but it doesn't seem to be lysing very well. I've used BugBuster before and I could see the cells clarify almost immediately and they use non-ionic detergent. Does anyone have any suggestion which detergents I should try and at what concentration? SDS is out as it's incompatible with IMAC resin.

[donny]

How about incubation time? I have to check if we have lysozyme though.

I read the information on B-Per and the detergents in there are removable by dialysis. That seems to imply that they are using octyl thioglucoside/glucoside and CHAPS/CHAPSO. Are these detergent any stronger than Triton-X?

[pDNA]

lysis time very much depends on the amount of cells you have in your pellet ...i would assume 15 min @RT should do the job.

No idea if octyl thioglucoside/glucoside and CHAPS/CHAPSO is a stronger detergent than Triton-X100, sorry!

Regards,
p

donny, on 13 February 2011 - 01:52 AM, said:

How about incubation time? I have to check if we have lysozyme though.

I read the information on B-Per and the detergents in there are removable by dialysis. That seems to imply that they are using octyl thioglucoside/glucoside and CHAPS/CHAPSO. Are these detergent any stronger than Triton-X?

[donny]

Ok, I've just tried the lysozyme lysis. Just as a trial, I lysed pelleted cells from 1mL overnight culture with 500uL 1mg/mL lysozyme in 20mM Na phosphate buffer, 0.1% triton x-100. Immediately, I see the suspension turn clumpy. Is that lysis or some kind of precipitation? There's no increased viscosity associated with genomic DNA. Am I doing it right?

Still curious about the composition of commercial detergent formulation or related recipe for cell lysis, if anyone has any suggestion.

[phage434]

B-Per uses octyl glucoside, according to the patent.

[proteaMatt]

Have you considered an acid labile surfactant or a cell lysis kit that uses one? I use it frequently because the E. coli lysates that I prepare are typically analyzed by MS and most detergents interfere with that. Downstream processing becomes much easier because you can cleave the surfactant by lowering the pH to 2.5 to 3 (with TFA or formic acid). I looked over the specs of the IMAC resin and they suggest using a 50 mM PBS buffer, which from my experience would easily overcome a pH of 3 and allow that resin to work effectively.

Edited by proteaMatt, 24 February 2011 - 07:03 AM.