The lab I work in has BioRad's Trans-Blot Turbo transfer device, but we'd like to use it with home-made buffers, gels and fibre pads as described in Luisa F Jimenez-Soto's protocol "Trans-Blot® Turbo™ Transfer with Home-made buffers" which is available on protocols.io .
While this protocol greatly increases the transfer's current to 1.3 A and lowers the voltage to sub 10 V, the gels always disintegrate during the transfer process. These 7.5 and 10% SDS-PAGE gels are made by mixing 1.5 M Tris (pH 8.8), 10% SDS, H2O, acrylamide and APS/TEMED and they have been used successfully for a long time in wet transfer. The transfer device doesn't heat up much during the transfer process
I was able to prevent the gel from disintegrating by not adjusting the pH of the anode and cathode buffers to what is stated in the aforementioned protocol, but this greatly increases the resistance and heat buildup in the system leading to low resolution compared to wet transfer. I've found no documented cases of such "gel disintegration" happening in semi-dry transfer so I'm stumped.
What is your transfer protocol? I have only came across a problem with the gel if I transfer longer than 15 minutes. I do not have this much disintegration though. I also use commercial gels 4-20% Norwex.
Skylar King My transfer protocol is almost exactly the same as the original by Luisa F Jimenez-Soto (Preprint Trans-Blot® Turbo™ Transfer with Home-made buffers v1
) and the only variation to the stack is the addition of one extra layer of WypAll fibre to each of the buffer stacks. So the sandwich consists of (from the bottom of the stack):After adding each layer, air is removed from them using a roller and additional buffer matching that layer is added onto said layer to keep it properly hydrated.
The transfer programme is currently 20 minutes long and runs at a consistent 1.3 A and maximum of 25 V, but really the voltage doesn't surpass 10 V. The only difference from the protocol that I've spotted is not using Bio-Rad's mini-protean gels, but as far as I know these are very similar to ordinary SDS-PAGE gels.
I'll try and lower the transfer time to 10 minutes to see if this can mitigate the disintegration while providing good transfer efficiency. Thank you for your help Skylar.
I think you could try 15 minutes. I use BioRad reagents and still increase the transfer time so the larger proteins can transfer. I also have a protocol using Towbin's buffer that I have gotten to work (adapted by a colleague), if you message me, I would be happy to send it to see if it could help. The Towbin's buffer protocol is much simpler than using 2 Anode buffers. I do not want to post it here since it is not my protocol. Hope this helps!
After further testing it was deemed that the transfer duration is simply too long for our standard SDS-PAGE gel. Transfers over 10 minutes lead to the gel contracting and forming a "bubble-like" texture which seemed to negatively affect the resolution of the bands. Transfer durations shorter than 10 minutes quite effectively prevent all disintegration, but predictably suffer from worse transfer efficiency of larger proteins so a change in buffer might be needed. It's of course surprising how there is such an incompatibility between the buffer and gel, but it is what it is.
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