Hello everybody
I had been doing semi-dry transfer in past few days and have experienced a persistent problem. I set-up my semi-dry transfer as follows:
1. Run a 12% SDS-PAGE gel under normal conditions.
2. I use a normally recommended transfer buffer made up of Tris, SDS and Methanol. I soak 3 pads and place them, then membrane, gel, 3 more pads above the gel. I take all the normal precautions such as no air gaps between membrane and protein.
I use a 0.2 micron membrane from Bio-Rad. Membrane is pre-activated with Methanol.
3. I have used to semi-dry transfer machines and used them at the following conditions : Constant Current -> 60-70mA and Voltage controlled so as to not cross 10V. Timing is generally kept between 30-40min.
I have observed that the low molecular weight bands ( 40-10kDa ) are passing through the membrane and found generally in the bottom-most pad. Next time, I also used two membranes placed one over the above and still the bottom bands pass through the membrane.
What can I do to prevent this ? Could it be the issue with the transfer buffer ?
Hello Abhishek Dubey
This should not be the case ideally. Maybe the time-current combination needs to be changed. I can suggest that you may refer to this paper from Dr. Y.P. Khasa's group: https://pubmed.ncbi.nlm.nih.gov/26631635/
His group deals a lot with protein overexpression and subsequent confirmation using WB (transfer using the semi-dry method). They make use of 1X western transfer buffer (Composition: 24 mM Trizma base; 192 mM glycine and 20% methanol) and a transfer time of 30 min/25 onto a methanol charged PVDF membrane (Millipore Corporation, USA). Hopefully, this will help you in your experiments. Good luck!
Hi Abhishek Dubey,
I would suggest optimizing the voltage and time duration to see which works for you (i.e., shorten the time with the current voltage, lower the voltage at the current time duration, or shorten the time with a lower voltage). Try to see what works best for your protein as it is at a lower MW. In the past, when conducting WBA for smaller proteins, I have used a wet transfer system and that worked well for me. If possible, try the wet transfer technique at a low temperature (i.e., ice-water bath or at 4 degrees C. Good luck!
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