Hello, everyone. I have a technical question about silver staining on LPS. Our samples comprise a heavier part of the O-antigen (often appearing as a smear) and three bands of much lighter lipid A and the core antigen. I have tried 10%, 12%, 12.5%, and 15% SDS-PAGE gels, but we still think the bands and smear were not separated very well. Should I try a higher percentage (like 18%) gel or optimize other aspects? Any input is welcome. Thank you so much for your attention and participation.
Yes to improve the separation :
You can increase the acrylamide in your SDS PAGE gels with 16% or 18%.
It is possible that you have an incomplete denaturation (try to heat your sample 5min at 80°C before depositing) or aggregates (additional filtration step 0.22µM or centrifugation may be necessary).
If the smears were not very well separated (presence of salts and large amount of protein loaded in the well) :
You can try to precipitate your protein before depositing with this protocol.
https://www.bioch.eu/methods/protocols/preparation-samples-sds-page-precipitation-acetone
Silver Staining Protocol for LPS
Tips: Avoid metal containers, protect silver nitrate from light, and stop development as soon as bands appear.
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