The purity of sodium carbonate is very important. We had same problem that was related to the sodium carbonate.

Is it completely clear? Because there should be at least some background. How long do you keep it in the developer? For a fixed time or until bands appear?

To Mr Nahed Hussien: I have done CBB and visualized the bands too

To Mr Tomas: Now i am keeping it till the band appears. Since the band is not appearing i sometimes left it for overnight also. i can see very meager amount of stain in the corners of gel

Sensitizer Sodium thiosulfate 20 mg in 100ml water incubated for 1 minute

Water wash three times each five minutes. Silver nitrate 200 mg in 100ml water + formaldehyde 100 microlitre incubated for 10min. Water wash three times each five minutes. Developer solution contains 2g of sodium carbonate + 25microlitre Sodium thiosulfate + 25 microlitre formaldehyde. Waited till the band appears but the gel has not uptake the developer soluton

Check your silver nitrate. Make sure that AgNO3 crystals are clearly transparent white in color. If there's a hint of pink or brown, your silver nitrate is bad and has to be replaced.

some times te water is the problem, try whit milli Q water.

To Konstantin i have checked it. It is fine

To Cay: I will try it

Mr Ruben let me start my next attempt by reducing the time duration and by increasing the sodium carbonate concentration

How much formaldehyde did u add to the gel?

Do you prepare your solutions freshly? Especially the sensitizer has to be fresh, because sodium thiosulphate decomposes over time.

One problem I have experienced with silver stain can be if your protein has a very low PI it may not stain. I was tearing my hair out as I can see my protein with a Bradford assay, but not on a gel. I eventually tried a hybrid protocol with silverstain and stainsall from the following paper, and it worked very well;

ANALYTICAL BIOCHEMISTRY 251, 227–233 (1997)

ARTICLE NO. AB972252

The Staining of Acidic Proteins on Polyacrylamide Gels:

Enhanced Sensitivity and Stability of ‘‘Stains-All’’ Staining

in Combination with Silver Nitrate

Harvey A. Goldberg1 and Kevin J. Warner

During my PhD-thesis some of my colleagues had also different problems with silver stains. Therefore, they tried alternative staining methods with comparable or better sensitivities. These methods (examples of two methods they used are listed below) led to very good results. Maybe you can also overcome your issues by trying these alternatives:

1. Donghoon Kang, Yong Song Gho, Myungkoo Suh, and Chulhun Kang, Highly Sensitive and Fast Protein Detection with Coomassie Brilliant Blue

in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Bull. Korean Chem. Soc. 2002, Vol. 23, No. 11, 1511-1512

-> higher sensitive than classical Coomassie G-250 staining (reported sensitivities: 100ng for classical Coomassie G-250 staining vs. ~30ng for the above mentioned method)

2. Giovanni Candiano, Maurizio Bruschi, Luca Musante, Laura Santucci, Gian Marco Ghiggeri, Barbara Carnemolla, Paola Orecchia, Luciano Zardi, Pier Giorgio Righetti, Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis, Electrophoresis 2004, 25, 1327–1333

-> sensitivity comparable or better than silver staining (reported sensitivities: 5-10ng for silver staining vs. 1-5ng for Blue silver staining)

By the way, both articles are available for free. Just google them ;-)

Mr Nitin overall I add 125 microlitre of formaldehyde

To Sandy: I prepare everything freshly

To Glen: This is new to me give me some days time i will work it out

To Benjamin: Sir i am getting band in CBB problem only with Silver staining

A useful guide:

https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314716762536/litdoc80634334AB_20110830181131.pdf

First of all check if you had loaded some quantity of protein, and if this is OK, check if formaldehyde reactive is ok and if the developer solution has the right concentration of it .

Here is the protocol I used. It's very important to shake your gel all the time and use the ultra pure water for solutions preparation. You need to prepare fresh solutions before your staining.

1. Fixation for 30 minutes in solution: 50 % (v / v) ethanol, 12 % (v / v) acetic acid.

2. Fixation for 30 minutes in solution: 10 % (v / v) ethanol, 5 % (v / v) acetic acid.

3. Shaking for 5 minutes in solution: K2Cr2O7 (3,4 mmol dm-3) and HNO3 (3,2 mmol dm-3). You have to prepare this fresh, just before you use it.

4. Washing the gel in ultra pure water: 3 times 10 minutes - until your gel becomes colourless.

5. Shaking for 20 minutes in AgNO3 solution (12 mmol dm-3). Prepare fresh and keep in the dark before you use it.

6. Shaking for couple of times in solution: Na2CO3 (0,28 mol dm-3), 0,01 % (v / v) formaldehide until brown precipitate forms.

7. Shaking in Na2CO3 (0,28 mol dm-3), 0,05 % (v / v) formaldehide until desired band intensity is achieved.

8. Stopping the reaction in 1 % (v / v) acetic acid.

I used to do this by shaking on rotary shaker and removing every solution by vacuum tube (you have to be careful not to suck up your gel :-), that's the good way to remove the most of your previous solution, before you add the next one.

I hope this helps.

Thanks everyone for your nice suggestion give some time for me to work out

I agree with Ramon. Perhaps the amount of protein is too low to detect. Can you run a positive control with enough detectable protein?

Hi,

you can try to optimize on sensitizing step in silver stain procedure, and the last step where you are going to stop the reaction. Consider you have loaded sufficient concentration of proteins.

You can try BioRad silver staining kit it worked well for me, did you see your protein standards?

I would strongly suggest you to check whether you have really used Na2CO3 (sodium carbonate) not NaHCO3 (sodium bicarbonate).

My be this protocol will be useful for you

Dear all,

I have identified this two reasons may cause problem in my staining technique

1. Silver nitrate

2. Protein concentration is very low in my sample

So wat i did is i have increased double the concentration of silver nitrate and volume of protein which gave me the result.

Thanks for everyone who gave me their valuable ideas

Hi Murugadas, I am experiencing similar troubles. I am very curious of whether the protein standards got stained when your sample amount was very low. In my case my ladders(prestained from Biorad) also showed no sign of reaction at all.

Dear Murugadas and Dear Charlie

There are different silver staining protocols but I use the following protocol reported by Blum et al., 1987. Electrophoresis 8:93-99:

1. Fixation: 50% methanol, 12% acetic acid, 0.5 ml/l of 37% p-formaldehyde; 60 min

2. Wash:

50% ethanol; 20 min

30% ethanol; 20 min

ddH2o, 2*20 sec

3. Sensitizing: 0.25 g/l sodium thiosulfate; 1 min

4. Wash: ddH2o, 3*20 sec

5. impregnate: 2 g/l silver nitrate and 0.75 ml/l of 37% p-formaldehyde; 20 min

6. Wash: ddH2o, 2*20 sec

7. Developing: 60 g/l sodium carbonate, 0.5 ml/l 37% p-formaldehyde and 4 mg/l sodium thiosulfate; Until appearing spots or bands

8. Wash: ddH2o, 2*2 min

9. Stop: 50% methanol and 12% acetic acid, 10 min

Please check the protein concentrations befor SDS-PAGE by using Bradford.

If you have any problems, please dont hesitate to contact me

Sincerely

The purity of sodium carbonate is very important. We had same problem that was related to the sodium carbonate.

Hi..

Checking the concentration of formaldehyde you are using also plays an important role .. 

Hi check with the concentration of your sensitizing agent. Increased concentration of sensitizer results in high level of background staining and also check your formaldehyde solution.