Semi-dry transfer conditions - Volts or Amps (Aug/11/2009 )
My question might be a bit silly but I thought I rather ask than make a silly mistake
In my old lab we had a Biorad's Trans-blot system and I always used 0.8mA/cm2 for 1h for transfer.
Now, in my new place, we have just bought a Novex Semi-dry blotter (currently waiting for it), and the manual says to use 20V for 30-60min.
Before reading the manual, I was just going to follow my "standard" protocol of 0.8mA/cm2 for 1h, but now I'm not sure what to do...
I would really appreciate your comments and suggestions on the matter.
I know in the end it'll all come to empirical determination, and I'll have to try different settings, just wondering whether to use constant voltage or constant amperage.... and what's really the difference and effects on the transfer of either.
Thanks in advance for your thoughts
I occasionally use the Bio-Rad Trans-blot SD for my transfers. For most proteins, I usually transfer at 15 V for 25 - 30 min as recommended by Bio-Rad (see below). So far I haven't had any problems. Although the Bio-Rad and Novex instructions don't seem to be that different (once again, see below) I think your best bet would be to follow the Novex instructions since they have probably optimized conditions for their apparatus.
From the Trans-Blot SD Semi-Dry Transfer Cell, Rev C Instruction Manual (Part 1703940)
Turn on the power supply. Transfer mini gels for 15–30 minutes at 10–15 V. Large gels
can be transferred for 30 minutes to 1 hour at 15–25 V. Do not exceed 25 V with this
instrument. A current limit (3 mA/cm2 for large gels; 5.5 mA/cm2 for mini gels) is
recommended to prevent excessive heating during the run. Under the strong fields
developed by this apparatus, transfers may not always be quantitative. A certain quantity
of protein may be transferred through the membrane and onto the filter paper below.
This manual can be found on the website. Bio-Rad
On the Trans-Blot SD Semi-Dry Transfer Cell page click on the document tab below the picture of the system. Then click on the Literature (downloads only) to find the manual.