Well, it is tricky. You should do the following:

1) Mix the Brillant blue with the water (for example 2 g of stain in 100 ml of water), stirring.

2) In between mix a 340 ml of Methanol + Phosphoric acid (35ml)+ 300ml of water

3) Add to the water/methanol/acid Ammonium sulfate slowly (wait for dissolve), and keep stirring until the salt will be completely dissolved (you should add water to reach 900 ml). After of that, keep stirring for 2 or 3 hours more.

4) Add the Water/brilliant blue to the methanol/water/acid/salt solution and keep stirring

5) Leave washout stirring overnight.

6) Filtered and use it

How much ammonium sulfate we are talking about

Dear Sharad, if I don't guess that you would like to stain Gels, I would like to point you to: http://cdn.preterhuman.net/texts/science_and_technology/nature_and_biology/Genetics/Proteomics%20in%20Practice%20-%20A%20Laboratory%20Manual%20of%20Proteome%20Analysis%20-%20Tom%20Naven.pdf

Reiner Westermeier, Tom Naven (Eds):

Proteomics in Practice: A laboratory manual of Proteome Analysis

© 2002 WILEY-VCH Verlag GmbH,

ISBN’s 3-527-30354-5 Hardcover 3-527-60017-5 (electronic)

go to page “259 of 329” (right margin display position to turn –drag down pages rapidly):

= real book page 233

find: STEP 4: Staining of the Gels

1 Colloidal Commassie Brilliant Blue Staining

Recipe for Stock solution A & B and how to proceed: on p.233 & 234

Hopefully this hint could be of help too solving your problem...Best regards and good luck

Hello again after a long period(!) of stagnation in this thread:

a) Thanks to Md Sanower Hossain (:-));

b) it would be interesting to see whether the original requester has any opinion now ("However, nobody out there dares to post a simple protocol explaining how to make a stable and reliable solution of the colloidal version of this dye. Does anyone have any solutions? I refuse to believe there isn't any out there").

c) two other possibilities out of my files (NB:cave (C) Copyright! Find two pdf's enclosed (see next RE-post)

best regards,

Wolfgang

Two other possibilities out of my files (NB:cave (C) Copyright!)

(no.1)

Regards,

Wolfgang

Two other possibilities out of my files (NB:cave (C) Copyright!)

(no.2)

Regards,

Wolfgang

Since some minute ago I found another posting thread concerning the matter, I would like to add:

"Regarding protein re-staining"

I have done Coomassie Brilliant Blue (CBB) staining for protein detection then destained the gel for removal of excess dye not bound to proteins.

Now I want to remove the dye bound to proteins to go for silver staining with same gel. But in the destaining solution the dye bound to proteins will not remove completely. Could you suggest any other opinions?

Mar 28, 2012 • Modified Apr 1, 2012 by the author

to be found @

http://www.researchgate.net/topic/Gel_Electrophoresis/post/Regarding_protein_staining

which also should point anybody in need to the TOPIC GelElectrophoresis @

http://www.researchgate.net/topic/Gel_Electrophoresis/

Regards, Wolfgang

Have you tried adding glacial acetic acid to the solution at 10% concentration V/V? Stirring overnight using a magnetic stirer should do the job then.

Dear Muneeb Faiq, thank you for adding your opinion / technical hint to try "adding glacial acid to (?)solution at 10% concentration v/v". Since the initial requetser Angel J Rivera never did reply or gave some more detailed information on the (C)BB-=(Coomassie) Brillant Blue)[polyacrylamide gel] staining (stock) solution he has in mind (there might be several variants) I'd guess one doesn't know how the addition of glacial acid should work anyway...Despite the original request has been posted 5 years ago (exactly 2013-03-19) NEW and practical approaches might have been published and may possess better properties to do the job (assuming it was protein staining of bands on polyacrylamide gel).

I've found quite recently another, modified technique of CBB-stain (most of the experts will know of) which is reported to be able to improve the sensitivity of detection from 10ng to about 1 ng protein/band.

It is an improvement published by KANG et al, 2002.

It is:

J Vis Exp. 2009; (30): 1431. Published online 2009 Aug 3. doi: 10.3791/1431

PMCID: PMC3149902 PMID: 19684561

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels, by Nadine Dyballa and Sabine Metzger, Biological Medical Research Center (BMFZ), University Duesseldorf.

For convenience only:

1) there are at least 3 or 4 versions of the article:

1a) PMC: @NCBI.nlm.nih.gov: cf: *+) : http_s://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149902/ (as html. *+) NB: before pasting into your browser, delete the "underline-character _ " in between http and s: http_s:// , otherwise (pasting the usual URL : "https://....", see as follows), one arrives at the version stored / offered in RESEARCH GATE repository (where the article is designated as CHAPTER 4 in

Katrin Marcus (ed.), Quantitative Methods in Proteomics, Methods in Molecular Biology, vol. 893, DOI 10.1007/978-1-61779-885-6_4, © Springer Science+Business Media, LLC 2012

{(24) Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels. Available from: https://www.researchgate.net/publication/26745741_Fast_and_Sensitive_Colloidal_Coomassie_G-250_Staining_for_Proteins_in_Polyacrylamide_Gels [accessed Jun 10 2018]. }

1b) Article Fast and Sensitive Colloidal Coomassie G-250 Staining for Pr...

1c) the Pdf, via the link as indicated in PMC: http_s://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149902/pdf/jove-30-1431.pdf

1d) http_s://www.jove.com/video/1431/fast-sensitive-colloidal-coomassie-g-250-staining-for-proteins *+) This version contains also the video [NB: A subscription to JoVE is required to view this article. You will only be able to see the first 20 seconds ] as well as Comments: 25 valuable Comments (mainly on the technique)(Requests and answers by authors)

2) Abstract (from the PMC version @ NB: *+) http_s://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149902

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.

Several improvements of the original Coomassie protocol (1) have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution (2), and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol (3). Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol (4). The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.

Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

Above mentioned

References ( from *+) : http_s://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149902/

Don't miss the interesting Comments on the technique, or/and the ingredients used: NB: *+) applies (seee above)

http_s://www.jove.com/video/1431/fast-sensitive-colloidal-coomassie-g-250-staining-for-proteins and Scroll down to the end of the article