Hi researchers,
for the first time i am going to do silver staining of SDS PAGE. can anyone suggest me a good protocol for doing that? Thanks in advance.
You can try this. I used it in my undergrad research:
1. The gel was kept in a fixing solution having 50% methanol, 12% acetic acid and rest water, under constant shaking.
2. The gel was washed thrice in distilled water (~5’ each).
3. The gel was sensitized in 0.005% sodium thiosulfate solution for ~30mins, in dark since the reaction is light sensitive (under constant shaking).
4. The gel was rinsed thrice in distilled water (~5’ each).
5. The gel was kept 0.1% silver nitrate solution for ~30mins, under constant shaking (reaction is light sensitive).
6. The gel was rinsed and then kept in a developing solution containing 2% sodium carbonate, 0.018% formaldehyde and rest water (with shaking), until the bands were visible.
7. 50mM EDTA solution was poured on the gel to stop the reaction.
You can then store the gel in 50% methanol or image it
Please find attached the protocol from Thermo. However if you are using a kit, I hope the protocol will be available along with that.
However for the quick staining you can refer this link.
http://www2.warwick.ac.uk/fac/sci/chemistry/research/blindauer/blindauergroup/lab_protocols/silver_stain/
Dear Kumar, I am working in Ecological and Environmental grounds. I have tried to answer your question. The following article may help you
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1971133/
http://stanxterm.aecom.yu.edu/wiki/index.php?page=Silver_staining_of_SDS_PAGE_gel
https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/protein-gel-stains.html
I recommend you to read the publications of Rabilloud. It's for 2D electrophoresis gel but it's the same for SDS PAGE.
Silver staining is used for sensitive detection of proteins separated by 1-D and 2-D SDS PAGE. Attached article lists a protocol of Silver staining. In the Guildlines paragraph, it points out something you should be aware of:
"Many silver staining protocols and commercial staining kits are not compatible with mass spectrometry due to the use of crosslinking reagents. Commercial silver staining kits compatible with mass spectrometry are available from different suppliers, including ProteoSilver Plus (Sigma) and Dodeca Silver Stain (BioRad). "
You can try this. I used it in my undergrad research:
1. The gel was kept in a fixing solution having 50% methanol, 12% acetic acid and rest water, under constant shaking.
2. The gel was washed thrice in distilled water (~5’ each).
3. The gel was sensitized in 0.005% sodium thiosulfate solution for ~30mins, in dark since the reaction is light sensitive (under constant shaking).
4. The gel was rinsed thrice in distilled water (~5’ each).
5. The gel was kept 0.1% silver nitrate solution for ~30mins, under constant shaking (reaction is light sensitive).
6. The gel was rinsed and then kept in a developing solution containing 2% sodium carbonate, 0.018% formaldehyde and rest water (with shaking), until the bands were visible.
7. 50mM EDTA solution was poured on the gel to stop the reaction.
You can then store the gel in 50% methanol or image it
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