protein stain, B-PER

From: Thomas Edwards
Date: 15 March 2012 14:24
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 from bacterial cells without harsh chemicals or mechanical procedures like sonication"), but would like to try a variety of similar things if possible. Any advice from the community out there? Anybody know what goes into B-PER or similar things (I know there's some Dnase and lysosyme in there – but which detergents are compatible with Ni, GST, how much do you need etc)??
 2.  Staining SDS gels. There are various concerns from lab members about safety re methanol in stains, microwaving stains etc etc. "Instant Blue" claims to have none of these problems.  Quote: "Protein gel staining takes around 15 minutes without the need to wash, fix, microwave or destain". But again, I'd like to try things to see if they work for us (before spending cash - yes, I am spending averse…!). Anybody any suggestions for quick, non-fix, non-methanol, non-microwave, non–destain protein gel stains? Have tried home made colloidal coomassie but our protocol still requires fixes and washes that made it not really worth while.

Happy to collate thoughts on replies offline and post summary.

Many thanks
Ed

T.A.Edwards Ph.D.

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From: Jacob Keller
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

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From: Cécile Breyton
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. Sensibility is as good (slightly better) than Coomassie.

The gel can be stored in H2O (but proteins diffuse after a while), destained for other staining (Coomassie, silver...), by incubation in 50 mM EDTA 5-10 min.

CuCl2 and EDTA solutions can be re-used.

Discard Cu in appropriate waste.

From Lee et al, 1987, Anal. Biochem 166, 308-312.

Cécile

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From: Ed Pozharski <epozh001@umaryland.edu>
Date: 15 March 2012 14:48
To: CCP4BB@jiscmail.ac.uk
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.
Temperature in the sonicated sample may go pretty high under regular
sonication protocol - we have the temperature probe and it easily goes
over room temperature.

I may certainly be wrong on both assumptions.  And French press would
suffer from the same heating problem - some pre-cool the whole thing in
the cold room, but I don't know how much it helps.

Cheers,

Ed.

--
"Hurry up before we all come back to our senses!"
                          Julian, King of Lemurs

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From: Jacob Keller 

Hey, that was my post! NB this cannot be used for native gels, I don't think.

Jacob

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From: Doug Ohlendorf
1.      We like Bugbuster. You can get it at 10X concentration so it is
fairly economical. We do add lysozyme and DNase as well.
2.      We use the NuPage gels and SimplyBlue SafeStain and it works very
well. We can run a gel in 20-30 min and have it stained and destained a hour
later using the microwave to heat the solutions. There is no methanol in the
procedure and gel runs at neutrality.

Doug

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From: Roger Rowlett

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 at capsicum.colgate.edu/chwiki.

Cheers,

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From: Artem Evdokimov
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 chromatography and (if no salt is added) for ion exchange primary capture. For the remaining 10% we try every other method under the sun. For gels - 15 minute biorad gels are very nice (have to have 300v power supply) and stain/destain in our hands is abou 5-15 minutes with a microwave. We destain in boiling water, with no other chemicals involved. Any coomassie based stin works well enough for this. Some ppl in our lab love the stain free gels but you have to remembr to count trp residues as it relies on trp chemistry to work.
In short, this (and a number of other tricks) makes it possible to have a three step purification done in one 8 hr work day.

Artem

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From: Kothe, Michael GZ/US

Hi Artem,

I have stayed away from these types of reagents in order to avoid detergents, and also because of anecdotal reports that these work less well for large-scale preps. Are you saying you use them routinely for preps of crystal-grade protein? Am I being too concerned about the presence of detergents?

Thanks,

Michael

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From: Claudia Binda
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 proteolysis, precipitation or instability
during purification (after sonication) behave very well using Emulsiflex.
It requires some maintenance but it solves many problems.

Claudia

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From: Federico Forneris
Hi,
if you use pLysS E coli strains and you're working with soluble cytoplasmic
proteins, a couple of cycles of freeze-thawing in LN2 will break your cells
pretty well with high protein recovery, without the need of extra exotic
chemicals added to your mixture. Just put some DNAse to avoid excess
viscosity of your sample and get rid of nonspecifically bound DNA. If the
cells are not pLysS, the same method works fine if you add to your lysis
buffer a bit of lysozyme and do a couple more cycles.

Regarding protein stain, here's my suggestion for a 5-minute clean,
efficient stain-destain protocol (with microwave, though):
- after running your gel, put it in a glass beaker or even better in one of
those low-profile glass containers used to make ice baths, filled with ~1 cm
of deionized water
- microwave the gel for 1 minute, max power (on my microwave, 1000W)
-wash out the water, then add 40 ml of PageBlue. Cover your container with a
paper towel (do not seal it), and microwave 3 times for 15 seconds at
maximum power (1000W), gently shaking the container between the cycles. The
blue stain should never boil. If you see that it starts boiling, reduce the
length of the cycles.
- collect back the blue stain solution, rinse the gel with water (again, 1
cm in the container), and microwave for 30 seconds. Change the water again
1-2 times. it's done, low background, ready for imaging.
In my hands, the whole procedure takes less than 5 minutes and gives
beautiful gels. The PageBlue solution that we use (Fermentas) is much more
sensitive than standard coomassie R250 and can be re-used an incredible
number of times. This makes this staining solution cheap despite the cost of
a 1L bottle (I am staining 3-4 gels per week and I've been using the same
50ml tube of PageBlue for the last 3 months at least). Without the
microwave, the same solution works well if you stain 10' and destain in
water for about 1 hour.

HTH,

Federico Forneris, PhD
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