Western blot transfer problem - (Jul/27/2006 )
Hi everyone,
I am having a problem with the transfer buffer for a western. Atleast I think it's a buffer problem. Even though I set the voltage to 70V, it runs between 50-60V, froths a lot and even stops in the middle. We checked the pH of the buffer solution and it is fine (8.3). Made fresh solution but we are still facing the same problem. Any suggestions?
for the transfer?
the voltage seems quite high.
Usually I do 150 mA, and it's around 18-20V.
In fact 0.8mA per square centimeters of gel is enough.
Hi,
Just to confirm if buffer is as follows: 25 mM Tris-base, 1.44% glycine and 20% methanol (optional).
Could try using other blotting apparatus to test the transfer with the prepared buffer. If there's no problem, it's probably the blotting apparatus.
Hi,
Just to confirm if buffer is as follows: 25 mM Tris-base, 1.44% glycine and 20% methanol (optional).
Could try using other blotting apparatus to test the transfer with the prepared buffer. If there's no problem, it's probably the blotting apparatus.
We are actually using 48mM Tris-base, 39mM glycine and 0.037% SDS along with 20% methanol. This is the recepie one of our post-docs has always used and it has worked for her. I will try with a transfer buffer that you suggest. Maybe SDS is the problem.
Thanks so much for your advice.
the voltage seems quite high.
Usually I do 150 mA, and it's around 18-20V.
In fact 0.8mA per square centimeters of gel is enough.
We have always done transfers at 70V or even at 80V and they have worked well. I will try using a lower voltage. How long do you transfer for?
Thanks for the reply.
for the transfer?
the voltage seems quite high.
Usually I do 150 mA, and it's around 18-20V.
In fact 0.8mA per square centimeters of gel is enough.
We have always done transfers at 70V or even at 80V and they have worked well. I will try using a lower voltage. How long do you transfer for?
Thanks for the reply.
I'm interested in proteins <50 kDa. One hour is enough.
For heavier proteins, you should increase the time of tranfer.
I am having a problem with the transfer buffer for a western. Atleast I think it's a buffer problem. Even though I set the voltage to 70V, it runs between 50-60V, froths a lot and even stops in the middle. We checked the pH of the buffer solution and it is fine (8.3). Made fresh solution but we are still facing the same problem. Any suggestions?
May try to rinse the transfer tank thoroughly with distilled water. Someone may not have clean them properly and salts and SDS built up.
I hope this may help.
Did you try to use another -a different model- power supply? It sounds like resistance is gradually increasing and transfer is stopping. We have two different models of power supplies with different current and voltage limits, and we also observed stopping of transfer or electrophoresis sometimes.