Hi,
I'm using a Biorad Mini-transblot electrophoresis apparatus to transfer proteins to nitro membranes. I'm using the cooling unit in the tank and a lot of ice around the tank. I'm using the Towbin buffer (tris-gly) with 20 % of MetOH and just one sandwich (pad, paper,gel, membrane, paper,pad) at time. I set the Voltage @ 100 V (constant). Which should be the expected mA for such conditions (single gel!)?
To which degree very low temps (I think I'm below +4°) could affect the current?
Do you know if membrane pores size affects the resistance of the sandwich and therefore the mA?
Thank you.
Hi Roberta,
Usually the rule is 0,8 to 1,2 mA /cm2 membrane. Mini-gels have 8,6 cm x 6,3 cm dimensions (54,18 cm2) . I cut my filter papers (in a semidry blot unit) and I take the 1,2 mA/cm2 as standard transfer, what makes a 64mA per blot. That for 1 hour 15 min (for proteins between 250 kDa and 25 kDa, but I am using PVDF membranes (0,22µm pores) so I don't loose my small proteins.
If you want to make sure that the protocol you choose is the right one for your protein, stain your gel after the transfer with coomassie. You can then see how much of the protein stayed in the gel.
Good luck!
Hi Luisa! Thank you very much for your reply.
My problem is that with the settings/conditions I'm using, according to Biorad manual/technical assistance and also my past experience, I should have an output of current of 350 mA for a single gel, but I barely reach 120-150 mA... Thus I was wondering whether very low temps or membrane pore size may affect the current output...
I've been using this system for years without any problem and with the right V/mA output, but now I'm setting up the system in a new lab that never used tank transfer before and I'm having problems...
Are you using the same buffer system? I agree with Wilco, 350 mA is too high for my experience.
But if you say you had the same before, try to see if something has changed in the buffer. The parameters that can change the Amperage (resistance) of the system are buffer (anode and cathode maybe in false area?), the filter and the gel.
The low temperature is just saving your gel from overcooking with so much voltage (my volts are only from 10-25V!)
good luck
Luisa
Hi! I eventually got 340 mA for 100 V as predicted by Biorad manual/tech assistance and as I observed in my past experience with tank transfer systems. I think it was because the last time paper/membrane were not perfectly fitting the gel size!
I have one more question...I've never used transferring buffers with SDS before...does anyone know if Ponceau Red staining works worse (poor staining) in presence of SDS?
Thank you again!
It doesn't matter if you use SDS or not. Put your membrane in MilliQ water immediately after transfer for a minute or so, and then soak in Ponceau S. It will stain allright.
Keep in mind that the more SDS you use, the less of your protein will bind to the membrane. This becomes apparent especially for low-molecular weight proteins.
First time I didn't rinse the membrane (you don't have to, if you don't use SDS in transfer buffer)...now Ponceau is working...thank you.
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