I used a 1.5mm thickness gel for western blot at 100v for 90min, but the transfer did not complete. Is it necessary to increase the time for an effective transfer?
Hello,
I usually transfer my blot just like you did, and it works good.
What is your transfer buffer?
And the size of your protein of interest?
If it is a big protein, increasing the transfer time may help. If it is a small protein, it has probably been transferred (supposing that when you say "not complete", you refer particularly to big proteins).
transfer buffer is Towbins buffer, a large amount of protein still remains in the gel
I use the same buffer.
What is the % of your gel and the size of the protein you want to study?
A 10% gel at 100V gets complete transfer (including proteins of 230kDa) in 2hr 30mins. If you reuse transfer buffer use ice packs to cool down the heat generated.
Make sure your buffer won't be old, and as Goodwin said try too keep it cool. check your transfer tank whether is working properly. In my lab we are doing a lot of WB and transfer condition was as yours but suddenly got problem and transfer was not complete. We took another tank and found out problem is tank itself.Also see if there is salt around the electrode in your tank and clean it.
Then your protein size is important higher molecular weight need longer time and vice versa.
Transfer is never complete.The most important fact is do you see your protein on your membrane after the western.High size proteins are difficult to transfer. The percentage of acrylamide is important. For this size of protein try to reduce the percentage. If you are at 10%, try 8% of acrylamide.
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