I am currently trying to transfer ~ 260kDa protein, using 1x transfer buffer with 0.1% SDS as recommended. I find that the protein ladder lanes disappear after 45 minutes and 2 hrs; the conditions I use are as follows: 7.5% gel, 350mA constant current. Can anyone help explain what is going on, whether the current is too high or is something else wrong with the protocol?
Hi,
I think the current is too high and try reducing time as well at the moment you doing 2h and 45 min, so would try 2h or 2h 30 min. I used 50V for 2h for 72kDa protein and works fine. Also you can try staining your gels afterwards to check if there is any protein still on the gel.
Good Luck..
Hello Sybil,
do you use a semi-dry or a wet-transfer system for blotting? For large proteins wet-transfer gives usually much better results.
Best regards
Which methanol concentration do you use?
Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will prevent precipitation of the (large) protein in the gel.
Hi Sybil, do you used ponceau solution to see your transfer protein? Do you see that also your protein, as the ladder were fainted?
In this case I also think is an amperage and overheat problems. it is better to use the constant voltage and to follow the advices about the overnight transfer.
If you do not have a cold room and you are afraid of overheat you can try to lower the voltage (or amperage if you prefer this) and transfer 2h and 30 min, control always the temperature of your buffer (touch the tank during the transfer, and control the buffer after the transfer). Check your band with ponceau and good luck!
Hi Sybil,
also you should checking pH of your stock solution.
for large proteins it is recommended to not use methanol in transfer buffer or at least reduse it to 10% of transfer buffer volume. Maybe your transfer should last longer (overnight) in cold conditions, like in a cold room, in a fridge ;P or with a cooler (if there is a place in your tank for that).
Recommend you this link from abcam
Keep finger crossed!
http://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf
I agree with advises of the other people, i advise you try to see also Isoelectric point of your protein... which kind of transfer buffer did you use ? if you are using TG buffer (pH 8.3) there is no problem because you used TGS (pH 8.3) for running. Otherwise if you used other transfer buffer at different pH (hight pH>10) it is possible that your protein precipitates in sds gel (pH 8.8-->10), for example as in my experience with proteins also of low molecular weight but with a very low isoelectric point (pH
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