I'm doing semi dry western blotting. I can see the bands right after the ponceau staining, but sometime it happens with my colleagues that bands were visible after the ponceau stain during washing with deionized water. Ponceau stain is ready made, and the membrane which we are using is nitrocellulose membrane (0.45um pore size). We used to take care of membrane activation before transfer, which means soaking into blotting buffer containing methanol, and after transfer on membrane, add ponceau sol directly without any washing in buffer. Could you suggest why sometime bands were only visible after ponceau stain? Can it be only because of less protein transfer on membrane?
Thanks…..
It must be related to your methanol step. When you destain with methanol, you denature your proteins, change their conformation and therefore little transfer. May I suggest that you destain your membrane (very carefully) in a laboratory microwave oven (not a food oven). Add some water to the membrane and place it in a small tray with enough water so your membrane does not dry. Add time in increments (do not "cook" your blot). You might be able to destain without using methanol. It is also faster and the integrity of the proteins is maintained. Hope this works.
Any PBST/TBST, PBS/TBS, whatever is middle salt (150mM) will destain you reversibly. Than after water wash you can do it again.
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