14 replies to this topic

[mcb56]

Hello, I have heard that when destaining a coomassie stained gel that it is good practice to put a curled up kim wipe or 2 with the gel in the solution. The idea being that the kimwipe soaks up the dye. This does not make any sense to me however. The dye seems to come off the gel and mix into the destain homogeneously. The kim wipe absorbs the solution as a whole, not just the dye in the solution. Therefore I can assume that the concentration of dye in the solution remains the same with or without the kimwipe and it should not make a difference if the kim wipe is in there. Am I correct?

Thank you.

[phage434]

No, Kimwipes are dyed in the same way that your bands are dyed.  The dye is captured by the (relatively) strong binding of the dye with the kimwipes, and the equilibrium concentration of dye in solution goes down.

[Gerard]

We use several several times a fresh destaining solution and the collected stained solution is regenerated by mixing with activ carbon and filtration. This diminished the wast more than 95% and lowered the costs considerably.

[Prep!]

we use fresh destaining once the old oe is colored enough.. luckily we need not recycle it!!!  
you can even try heating the gel with destaining once or twice... but there is a chance of protein loss.. specifically low molecular weight ones.. and also dont heat if your acrylamide percent is  low!!!  

[K.B.]

I use "home-made" colloidal Coomassie for staining (freshly prepared for important gels, used for non-important ones) and distilled water for destaining - even cheaper.

[Prep!]

yeah colloidal also works well with less background... there are lots of different recopies to make it though.. which one do u use K.B?

[K.B.]

"Blue Silver" (source DOI - 10.1002/elps.200305844 )

working solution:
0.12% CBB
10% phosphoric acid
10% ammonium sulphate
20% methanol

stock solution (without methanol):
0.15% CBB
12.5% phosphoric acid
12.5% ammonium sulphate

Dissolve 125g ammonium sulphate and 147 mL of phosphoric acid (85% conc.) in approx. 500-700 mL of DI water. Disperse/dissolve 1,5 g CBB in small amount of water and add to solution. Stir/shake vigorously for at least 1 h (I often leave it stirring for the night). Fill up to 1 L with DI water. Mix 4:1 with methanol before use (shake).

[Prep!]

thanx there i guess the CBB is G 250 and not R 250 right?!!

[K.B.]

Right

[Prep!]

K.B., on May 17 2010, 07:22 PM, said:

Right

thank kb but i have a slight doubt here.. how did u come up with 147 ml for 12.5% phophoric acid? if i understand right as it is 85% pure u have to add 15% more... and the mol wt is 98... so the onl;y thing remains is the density which might be very very different from what i get!!!
please correct me if tere is an error!!!

yeah i think you have ot considered density in the calculation??!! is it deliberate??!!! i mean it is v/v and not w/v is it??

Edited by Prep!, 17 May 2010 - 08:20 PM.

[K.B.]

Type of concentration (ie. v/v, w/v or w/w) is not stated in the article and I assumed (for simplicity) that it is v/v.

I need 12.5% which is 125 mL of "pure acid" per 1000 mL

85% acid is 80 mL of "pure acid" per 100 mL

125 - x
80 - 100
---
(125*100)/80 = approx. 147

[Prep!]

yeah i assumed that this is what you would have done!!!  

[K.B.]

(typo - there should be 85 not 80 in ratio calculation)

[Prep!]

K.B., on May 19 2010, 02:22 PM, said:

(typo - there should be 85 not 80 in ratio calculation)

yeah!!!  
actually i will be doing a silver coomassie and a colloidal direct comparision in a day or two.. will let u know of the results!!!  

[HomeBrew]

See also Dyballa N, Metzger S. 2009. Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels. J Vis Exp. Aug 3;(30):1431. (PMID: 19684561, here) and Pink M, Verma N, Rettenmeier AW, Schmitz-Spanke S. 2010. CBB staining protocol with higher sensitivity and mass spectrometric compatibility. Electrophoresis. Jan;31(4):593-8. (PMID: 20162584, here).