Western blotting with room temperature buffers - protocol says NOT to use cold buffers (Dec/14/2006 )
I just got the Tris Tricine Express Endurance SP precast gels from ISC Express and I'm planning to do a western blot for a small (8kDa) protein with PVDF. In the protocol that came with the gels, it says NOT to use precooled transfer buffer but to simply equilibrate the gels in transfer buffer for 5 minutes.
The transfer buffer is Tris Bicine. I am using the Bio-Rad mini protean 3 system and I'm wondering if I should put the little ice block in during transfer.
Without the ice block, the transfer buffer will heat up. The ice block will keep the transfer buffer closer to room temp during transfer, so you should still use it.
I agree with Zona Pellucida.
The protocol explicitly says "DO NOT cool transfer buffer" which is really odd. Later on it says to put the apparatus in ice bath for cooling after assembling sandwich and filling apparatus. The manual is also for 2 other precast gels that have different buffer system and requires cold buffer. It is not clear in the directions whether ice bath/block is ok for all 3 gel systems.
It just seems like electrical shock hazard to but it in an ice bath, I'm going to go with ice block and transfer at 40V for 2 hr which was recommended.
It just seems like electrical shock hazard to but it in an ice bath, I'm going to go with ice block and transfer at 40V for 2 hr which was recommended.
the protocol was referring to the soaking step. maybe they were worried about crystallizing sds with cold buffer (reducing the efficiency of the soak in removing sds from the gel). they don't have a problem with cooling during the transfer itself. so either use the ice block or immerse your apparatus in an ice bath (not for semi-dry). there should be no shock hazard as long as the ice (and water) doesn't cover the top of the apparatus.
The transfer buffer is Tris Bicine. I am using the Bio-Rad mini protean 3 system and I'm wondering if I should put the little ice block in during transfer.
I agree with mdfenko: risk of crystalization of SDS
I used to transfer in cooled transfer buffer, with an ice block.
Once I forgot to chill the buffer. I transfered with RT buffer, with ice block : my best transfer. Now I always use room temperature buffer, with ice block. It's not too cold, and it avoids heating.
