Coomassie is much faster but 10-100XX less sensitive than silver stain. If your band is faint you should use silver, but its also a bit more tedious.

Coomassie is much faster but 10-100XX less sensitive than silver stain. If your band is faint you should use silver, but its also a bit more tedious.

Dear Surya Prasad Tiwari

Have a look on some articles, pdf, protocol and troubleshooting I keep them in my Lab.

Pls. find the attached files

https://www.chem.ufl.edu/wp-content/uploads/sites/17/2014/05/SDS-PAGE-Stain-Protocol.pdf

http://bitesizebio.com/9386/silver-or-blue-whats-the-best-stain-for-my-proteins/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1971133/

https://www.nationaldiagnostics.com/electrophoresis/article/staining-protein-gels-coomassie-blue

http://web.stanford.edu/group/chenlab/cgi-bin/wordpress2/wp-content/themes/chen/assets/docs/SilverStain.pdf

https://www.nationaldiagnostics.com/electrophoresis/article/coomassie-blue-stain-troubleshooting

http://onlinelibrary.wiley.com/doi/10.1002/elps.1150090106/full

http://aldrin.tripod.com/index-3.html

http://www.cytographica.com/lab/protocols/gel_destain.html

http://www.ruf.rice.edu/~bioslabs/studies/sds-page/sdsgoofs.html

Hoping this will be helpful

Regards

Prof. Houda Kawas

I think, you should go for silver staining.

You could try Comassie coloidal before silver stain.

After that you could try this protocol for silver stain:

4. Procedure:

Note: For each gel, add approximately 100mL of each solution, and stir gently.

This note, applies to points 4.1, 4.2, 4.3 and 4.4.

4.1. Fixation

4.1.1. Incubate the gels in acetic acid 10% (v/v) and ethanol 30% (v/v) for 30 minutes. Repeat

this procedure two more times;

4.1.2. Wash the gels for 10 minutes in ethanol 20% (v/v);

4.1.3. Wash the gels for 10 minutes in MiliQ water.

4.2. Sensitizing

Note: If there is no stock solution of sodium thiosulfate, prepare one of 1M:

- Weight 0,39g of pentahydrate sodium thiosulfate for 1mL of solution;

In step 4.2.2, use 80.6 μL of this solution for 100mL of sensitizing solution. In step 4.4.1,

use 4 μL for 100 mL of developer solution.

4.2.1. Incubate the gels, for 1 minute, in sodium thiosulfate (0,2 g/L);

4.2.2. Wash the gels during 20 seconds in MiliQ water. Repeat this procedure two more

times.

4.3. Coloration

4.3.1. Incubate the gels for 30 minutes, in a silver nitrate solution (2 g/L);

4.3.2. Wash the gels with distilled water for 10 seconds.

Note: The silver nitrate solution is prepared in that moment and must be protected from

light with aluminium sheets. After it’s used, pour the solution into a toxic waste flask.

4.4. Revelation

4.4.1. Incubate the gels within a 37% formaldehyde solution (0,7 ml/L), sodium or potassium

carbonate (30 g/L) and sodium thiosulfate (10 mL/L).

Note: Formaldehyde solution – add 70 μL for each 100 mL of solution.

Sodium thiosulfate - 1,6 mg of the 5-hidrated compound for 100mL.

The incubation it’s carried out for the time necessary until we get the band intensity

pretended.

4.4.2. Stop the revelation, incubate the gels within a 5% Tris base solution (m/v) and 2,5%

acetic acid (v/v) for 1 minute.

4.4.3. Store the gels in distilled water.

Dear

Also

Silver staining is better. Why? 3 nano gram of protein is detecting in SDS-PAGE. 

Dear everyone, Many thanks for your constructive ideas, interesting papers and protocols. First, I'll study thoroughly and adapt in my experiment. I'll get back to you soon. Thank you.

With best regards,

Surya Prasad Tiwari, University of Kiel, Germany

You most welcome

Houda

Remember that silver staining has a very small range were staining is linear with the protein concentration. If you want to compare different bands (or quantify them) silver staining it’s not the best technique.

Surya,  I see everyone has given you lots of material to read and protocols to follow.  Please remember that you have to control you experiment.  Just remember that you select the appropriate stain by the quantity of protein that you add to each well.  So when you set up your gel you need to know what concentration of protein is in your sample, example for Coomassie Blue you need 10 to 20 micrograms per well.  If it is a pure protein you would need much less.  If you do a silver stain start with 100 or so picograms.  If your sample is precious work your gel system out using something readily available like serum or commercial protein standard.  Good luck. 

Silver nitrate is better staining (SDS-PPAGE)

You will simply have to balance the tradeoff. CBB is easy and quick but Silver staining is far sensitive. So if you have good amount of protein, it will be better to use CBB but with smaller concentrations, silver stain will be an obvious choice.

The preferable staining is according to the amount of proteins. if the concentration of protein is lesser then you should use silver staining it is approx 100 times more sensitive than CBB, but it is time dependent and need to follow proper protocol. if the concentration is sufficient then you can use CBB, it is easy to use.