I use 100V and 60min for transfer step. The buffer is Towbin buffer containing 20% methanol. It’s already two times that I got the result as seen in the photo. One ladder seems fine but the other protein ladder is disrupted like that. Can anyone give me the suggestions and solutions please?🙏
This, to me, looks like an air bubble.
Looks like your transfer apparatus is performed in a tank and not using a semi-dry apparatus. Since you did this twice already, does this problem occur on the same side each time or is the location of this problem variable?
Check to make sure the clamp that holds your transfer stack together is tight and compresses the transfer stack efficiently. If the problem occurs on the same side, using the same equipment, then it would be a clamp issue. If variable, then make sure you are rolling out the stack well to avoid any air bubbles.
Alternatively, the transfer may be getting too warm, causing air to expand on that side, resulting in a poor transfer. If it is this, then place the tank in the cold room and try again.
Since the other ladder transferred fine, I don't think this is a sample issue. Check you equipment and/or transfer stack building method.
Kyle Skalenko Thank you very much for your advice and suggestion. I’ll make sure the clamp is tight enough.
It happened on the same side. I put icepack in the tank but in the last 20 minutes, the voltage decreased till 85V. So, I ran for another 20 minutes as I’m concerned the protein transfer was not complete.
No problem, Sandy Kha
Yeah, definitely check your equipment. I suspect that is your issue.
To check proper protein transfer, you can stain the membrane with Ponceau S. The stain can easily be removed with water (https://med.wmich.edu/sites/default/files/Ponceau_Stain.pdf).
You can stain the gel with Coomassie.
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