Re: [ccp4bb] protein stain, B-PER

ubject: [ccp4bb] protein stain, B-PER Dear BB, Apologies for being mildly off topic. Maybe. 1. We are trying to express (in E. coli) a protein which appears to be quite sensitive to mechanical disruption. We have ordered some B-PER (Pierce - "B-PER Bacterial Protein Extraction Reagents are

Re: [ccp4bb] protein stain, B-PER

Hi, we also stain gels in microwave: Comassie with 5% acetic acid to stain and just water for destaining. We keep the microwave under the fan to avoid breathing acetic acid. For E. coli cell disruption we are quite happy with Emulsiflex C3 by Avestin. In many cases proteins that undergo proteolys

Re: [ccp4bb] protein stain, B-PER

of detergents? Thanks, Michael From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Artem Evdokimov Sent: Thursday, March 15, 2012 7:04 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein stain, B-PER Hi :-) These subjects come

Re: [ccp4bb] protein stain, B-PER

Hi :-) These subjects come up regularly! Basically, in brief we use a nearly magical mix of bper and yper in 3 to 1 ratio, supplemented with salt, bensonaze and lysozyme. In my fairly extensive experience this works for about 85 to 90 % of cytoplasmic proteins with no issues for affnity chromatogr

Re: [ccp4bb] protein stain, B-PER

We like using a BeadBeater (Biospec) to disrupt cells. It is actually quite gentle, highly efficient, and very cost-effective. No potentially harmful sound or aerosols. For gel staining we use a low-toxicity ethanol-based stain-destain system. See our protocol wiki

Re: [ccp4bb] protein stain, B-PER

P4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein stain, B-PER Dear BB, Apologies for being mildly off topic. Maybe. 1. We are trying to express (in E. coli) a protein which appears to be quite sensitive to mechanical disruption. We have ordered some B-PER (Pierce - "B-PER Bacterial Protein Extraction Rea

Re: [ccp4bb] protein stain, B-PER

Hey, that was my post! NB this cannot be used for native gels, I don't think. Jacob On Thu, Mar 15, 2012 at 9:47 AM, Cécile Breyton wrote: > Hi Ed, > > A few years back there has been a thread on copper staining that leads to > "negative staining", but works well, is very fast and simple. > > -

Re: [ccp4bb] protein stain, B-PER

By mechanical disruption you mean sonication only or have you tried the French press? Assuming that you use sonication, and assuming that you follow a fairly standard protocol (e.g. something like 10sec pulse/20sec pause on ice for 3 minutes total), it may be heat not ultrasound that gets to it. T

Re: [ccp4bb] protein stain, B-PER

Hi Ed, A few years back there has been a thread on copper staining that leads to "negative staining", but works well, is very fast and simple. - Rinse gel 10-30s in H2O - incubate in 300 mM CuCl2. The gel stains in 3-5 min: background becomes opaque, the protein bands remain clear. Sensibilit

Re: [ccp4bb] protein stain, B-PER

Switch ethanol in the stain. Also, there was talk a while ago about making your own "instant blue" stains--it works like bradford reagent, I think. Perhaps look back at previous postings? JPK On Thu, Mar 15, 2012 at 9:24 AM, Thomas Edwards wrote: > Dear BB, > > Apologies for being mildly off top

[ccp4bb] protein stain, B-PER

Dear BB, Apologies for being mildly off topic. Maybe. 1. We are trying to express (in E. coli) a protein which appears to be quite sensitive to mechanical disruption. We have ordered some B-PER (Pierce - "B-PER Bacterial Protein Extraction Reagents are designed to extract soluble protein fr