Hi Arkadiusz,

You probably asked two different questions and I think the Coomassie Blue (CB) staining and home made recipe for protein molecular weight marker for Western blots are two different issues.

For the first part of your question I guess you wanted to stain proteins in solution with CB not staining proteins in gel with a CB solution. Can I ask you a question? What is the usefulness of staining proteins in solution with CB? If the purpose is just to detect the presence of proteins there are other easy conventional ways/tools available which would serve your purpose like UV scanning at 280nm, Lowry, Bradford or BCA assay. If you want to stain protein is PAGE you can have the protocol elsewhere. It's one of the most used methods.

To answer your second question I would say the question is not well formulated. What molecular wt region you are looking for? This will determine the MW standards of your interest. Prestained and unstained good and reliable MW standards are available commercially that can be used for SDS -PAGE and for WB after electrophorectic transfer to NCP or PVDF. Still if you want to formulate a home recipe you can definitely do this by adding known molecular wt proteins in the desired ratio (I am not sure about the proportions). For identifying proteins in the broader region 7.6-216 kDa.

Aprotinin, Lysozyme, Soybean trypsin inhibitor, Carbonic anhydrase, BSA, β-Galactosidase and myosin are usually used used. To get a detailed description of the respective mol wts and the expected positions of the bands in SDS-PAGE you can consider following link for further reading.

http://www.bio-rad.com/en-us/product/prestained-sds-page-standards

In this link go to " Description" page.

But you have to be cautious depending upon the conditions of gels used for electrophoresis (Tris-Hcl, Bis-Tris or Tris-Acetate) and also the % of gel formulation could dictate you selecting right standards for your purpose. To give yo a brief idea if am sharing a file as ready reference.

Hope that helps!!!!

I hope the attached paper can help you with it.

Cheers.

Hi Arkadiusz,

You probably asked two different questions and I think the Coomassie Blue (CB) staining and home made recipe for protein molecular weight marker for Western blots are two different issues.

For the first part of your question I guess you wanted to stain proteins in solution with CB not staining proteins in gel with a CB solution. Can I ask you a question? What is the usefulness of staining proteins in solution with CB? If the purpose is just to detect the presence of proteins there are other easy conventional ways/tools available which would serve your purpose like UV scanning at 280nm, Lowry, Bradford or BCA assay. If you want to stain protein is PAGE you can have the protocol elsewhere. It's one of the most used methods.

To answer your second question I would say the question is not well formulated. What molecular wt region you are looking for? This will determine the MW standards of your interest. Prestained and unstained good and reliable MW standards are available commercially that can be used for SDS -PAGE and for WB after electrophorectic transfer to NCP or PVDF. Still if you want to formulate a home recipe you can definitely do this by adding known molecular wt proteins in the desired ratio (I am not sure about the proportions). For identifying proteins in the broader region 7.6-216 kDa.

Aprotinin, Lysozyme, Soybean trypsin inhibitor, Carbonic anhydrase, BSA, β-Galactosidase and myosin are usually used used. To get a detailed description of the respective mol wts and the expected positions of the bands in SDS-PAGE you can consider following link for further reading.

http://www.bio-rad.com/en-us/product/prestained-sds-page-standards

In this link go to " Description" page.

But you have to be cautious depending upon the conditions of gels used for electrophoresis (Tris-Hcl, Bis-Tris or Tris-Acetate) and also the % of gel formulation could dictate you selecting right standards for your purpose. To give yo a brief idea if am sharing a file as ready reference.

Hope that helps!!!!

Hi Amritlal,

I'm sorry for not being clear with my question. In fact the reason for my question is a difference in the price of commercially available prestained protein ladders and those that are just a mixture of unstained proteins . I was wondering if I can stain myself the proteins in solution to have them visible in SDS-PAGE and WB. My first thougth was to use Bradford reagent for staining proteins and then put them into Laemmli loading buffer. What do you think about the idea?

Anyhow thank you for all those interesting points you raised,

all the best,

Arek

Hi Arkadiusz,

Now I clearly get the sense of the question that you have asked. I must say that you definitely have a novel and interesting idea which could be cost effective. But how to purify the CB stained proteins to be used as pre-stained MW standards? You definitely do not want to run a bulk of CB solution in your SDS-PAGE along with MW standards. All proteins even do not interact similar way towards CB (http://www.jbc.org/content/260/18/9976.full.pdf). If you stain and purify proteins in solution I personally do not know whether the CB stained individual marker proteins will retain the stain after SDS-PAGE is run.

The pre-stained markers (161-0318) available from Bio-Rad do not show any different color that can be distinguished by naked eye as it comes solubilized in intense blue 1X Laemmli buffer (ready to load) that contain bromophenol blue. If by any means you are able to stain and purify MW standards still you have to calibrate those using known standards as the electrophoretic mobility could be different for the unstained and stained proteins.

If I find any further info I will definitely share with you.

Good luck!!!!

Hello Arkadiusz,

The most accurate MW of the protein marker(s) for SDS-PAGE are when they are "not stained". I don't know what exact end-point of your experiment is, hoverer, at the moment I don't see the rational in spending time for pre-staining your home-made markers for PAGE and/or WB, since you can easily (and most accurately) visualize your home-made markers by staining the gel with CB after completion of the PAGE. Alternatively, the marker bands on the membranes (both PVDF and NC used for WB) can be stained, marked and de-stained using inexpensive water-based Ponceau S staining. Hope it will help, and good luck...

@ Arkadiusz,

I also frequently use Ponceau S before blocking and right after electrophoretic transfer to check the transfer efficiency and to make it sure that the target molecular wt protein regions have been successfully transferred onto the membrane. As Gediminas mentioned either staining the gel with CB or staining the NCP/PVDF with Ponceau S could be inexpensive and less labor intense compared to the involved project what you are thinking of.

I also recommend to not stain the protein(s) you would like to use a reference... by staining you will increase the molecular weight. I am using also Ponceau S staining steps as mention before. Good Luck in your research !