Hello,
I looked for a principle for the transfer procedure.
I know that it depends on your instrument, buffer, Mw of proteins and the size of paper.
I prefer to use constant ampere due to the similar mobility of all proteins (low or high MW) and having a defined formula: 0.8mA/cm2 for semi-dry transfer.
As far as the size of PVDF(. 0.45µm) and gel were 40cm2, I run transfer with constant 0.05A for 40 min. unfortunately, proteins remain in the gel and did not transfer well.
The second time, I run with constant .1A for 1hour. during this, the voltage increase to 80v and then decreased. the efficiency of transfer looks good based on the pre-stained ladder.
When I repeated this procedure with the same buffer, current and instrument (Trans-Blot® SD Semi-Dry Transfer Cell) the voltage comes up to 110v and then decreased!!!!! The ladder passed the PVDF paper and reached to pad. I'll appreciate it if someone can help me?
1- What's wrong with my work? I expect that when I use constant ampere, the variation of voltage be similar(increase up to 80v) in same condition.
2- Why we shouldn't exceed 25v? how some one use voltage more than 25v?
Mohammadrasul Zareinejad I can partly answer on the 1st question. When I use constant value of ampere, voltage can be really different in same condition. It dependes on very slight differences in buffer (in my SDS-PAGE, for example), and voltage is changing bacause of different resistance.
You are using the same system I have been trying to standardize, because I usually use wet transfer. So, here are my recent findings.
1. I prefer to use voltage. The semi-dry system recommends to use 15 to 25V at different times for mini gels. The best time I found was 25V at 1 hour, using the Towbin buffer (25 mM Tris, 192 mM glycine, pH 8.3, 20% methanol), it transferred quite well, however I stained the gel and still many protein could be detected. (I attach evidence, I loaded 15 and 30ug of protein!) If your protein of interest has high molecular weight, you may add SDS to the buffer to ensure its capture in the PVDF membrane.
I also tried 25V - 30mins, it worked well too. And 15V -1h (that was didn't work). So, I highly recommend you 25V for 1h.
2. You can't use more than 25V in the semi-dry transfer because it could burn and damage the cathode. Have in mind that you can't also exceed 1 hour and 15 minutes of transfer because it can also affect the cathode and burn both your membrane and gel. So be careful with these parameters.
I hope this works for you too!
In a semi-dry system, you have a limited supply of ions to support conductivity. The Ohmian heat is distributed only in that small volume, causing high temperatures. In my experience, tank blotters give better results than semi-dry, largely probably for these reasons. The only reason to use semi-dry systems is when you want different buffers at the gel and membrane side (doi:10.1006/abio.1993.1313).
In addition, I use Dunn's rather than Towbin's transfer buffer (10 mM NaHCO3, 3 mM Na2CO3, for large proteins + 50 µM SDS, doi:10.1016/0003-2697(86)90207-1). It's not only better, but cheaper too. I work with 40 V for 1 h, resulting in a completely clear gel when I stain it with CBB.
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