I always blot overnight 10 volt and then 1hr - 100 volt but the signal is always so weak ... any ideas?
Do you run at room temperature or in the cold room? I would suggest overnight transfers to be performed in a cold room setting. However, i am not familiar with the MW of p-ATM, so i would suggest to stain your WB after transfer with Ponceau S stain as well as your gel after transfer to see if the transfer was sufficient or the protein was over transferred and went throw the membrane (hence MW is needed to provide a better idea on transfer time and needed voltage)
Bassam, the ATM MW is 320 kDa ..... and the blotting is in a cool room and we blott the membrane overnight 10 v
Based on the MW, i agree with Florian and Yang voltage anywhere between 25-30 should be suffecient. But still recommend the post WB transfer staining at least once to make sure it went well, best of luck!
A good trick I learned for long-time blots, is to use double or even triple membranes (stuck together, pre-treated, etc.). After the blot, you control-stain (Poncaeu S) each to ensure the transfer happened completely - on 1st, or 2nd membrane.
Florian, only for making it clear for me, I will blot overnight 25-30 V and then on the 2 nd day blot 100 V for 30 minutes or only 25-30 V?
Hamed, what kind of transfer are you going to use? Regular or semi-dry?
I would try 40V and (like Marcin said) use 3 membranes.
I always run at 50 V using PVDF, find it a lot better than nitrocellulose.
We do it like this. Blotting in the cold room at 50mA overnight.
I agree with Tatjana Heidebrecht, I recommend carrying out your blotting at 40-50 mA overnight in the cold room and the next day, continuing it for 2 h at 200 mA at the same circumstance.
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