Good day, I recently did western blot up until the transfer part, However, the proteins were not transfer from the gel to the PVDF membrane. The staining of Laemli buffer and Kaleidoscope were not found in the gel neither in the PVDF membrane. I stained the membrane with Ponceau S to validate if the proteins were in the membrane and nothing was transferred. I suspect that it was the parameters that I added for the transfer. Parameters were:
-160 Voltage
-0.4 Amperes
-1 hour
-Cold temperature
Transfer buffer contained:
20% Methanol and 10% 1X TGS diluted with distilled water.
Thank you.
- There are two systems for blotting, semi-dry and tank. In a semi-dry system the gel/membrane/filter-paper sandwich lies between two graphite plates, that serve as electrodes. In a tank system, the sandwich is placed in a tank with buffer, the much larger buffer volume limits heating, hence transfer is usually better (exception: for small proteins (< 10 kDa) the semi-dry system allows to use different buffers for gel and membrane, this can improve transfer).
- There are two different membranes that are used for protein blotting, nitrocellulose and PVDF. Of these, PVDF has the higher protein binding capacity and affinity, it also is resistant to fragmentation during the many incubation steps in western blotting. However, it is more expensive than NC.
- There are two well known buffer systems for blotting, Towbin (doi:10.1073/pnas.76.9.4350) and Dunn (doi:10.1016/0003-2697(86)90207-1). Dunn buffer (10 mM NaHCO3, 3 mM Na2CO3, for large proteins add 50 µM SDS) is cheaper and more effective than Towbin.
With a tank blotter and Dunn buffer I use 30 V (resulting in about 200 mA current) for 1 h. Make sure you use the ice-block that comes with the tank to prevent unecessary heating. Power supplies for 30 V that can deliver one or two Amperes can be obtained cheaply from electronics suppliers, then the (much more expensive) high voltage supply is kept free for electrophoresis.
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