hello every body!
I am transferring proteins on a 45 um NC membrane. when working with 90 volts for 100 mins, complete transfer in not achieved, as bands can be seen on the gel when stained with coomassie blue. is it normal? I load 30 ug of protein per well( 100 volts for 20 mins and then 130 volts for 70 mins).
when working with the same voltage for 150 mins, it looks like that proteins just go through the membrane as I can see the stained ladder on both sides of the membrane and no signal on the membrane. I'm confused. Isn't it too high for transferring protein for 2 hours on 90 volts?
your wise answers will be appreciated.
I'm old-school and subscribe to the mantra of lower, longer, colder... meaning 30V @ 90mA overnight in the cold room. This technique is slow, but I get full transfer to the membrane, i.e. little to no coomassie blue staining on the gel. I also use PVDF membranes. Newer technology of rapid transfers are available, but require purchase of kit specific sandwiches/ machines, which may be cost prohibitive, however they are much faster.
Dear Zabi,
I transfer the proteins on Nitrobind Cast, Pure Nitrocellulose membrane (Cat no#EP2HY00010, GVS Maine) with 175 volts or 300 mAmp for 75-90 minutes depending on the size of protein. I also make sure that transfer buffer is cold and I carry the transfer in cold room or you can put your transfer assembly surrounded by ice to keep it cold.
Hope this helps.
Sincerely,
Manisha
I'm old-school and subscribe to the mantra of lower, longer, colder... meaning 30V @ 90mA overnight in the cold room. This technique is slow, but I get full transfer to the membrane, i.e. little to no coomassie blue staining on the gel. I also use PVDF membranes. Newer technology of rapid transfers are available, but require purchase of kit specific sandwiches/ machines, which may be cost prohibitive, however they are much faster.
40v overnight (12-15hrs) is my choice for complete transfer especially for proteins above 100 kDa. I leave the set up at room temperature (only new buffer...no reuse of buffers for room temp) only with a ice pack inside the wet transfer apparatus (Bio-Rad). Note: reusing buffers offer more resistance and generate more heat.
if you worriing about over-transfer, use double membrane to check it. colored markers usually are visible from both side of the membrane, cause membrane is transparent a bit, when wet. use appropiate setup depends on your target MW mostly (detailed above).
We often probe for proteins 15-180 kd and transfer 100V 90min. I haven't had an issue with proteins transferring through, but also have no protein left in the gel. You might want to check your buffer solution and make sure that it is properly maintaining power, or borrow another power supply and make sure you power supply is running well.
By the help of a cool transfer buffer and putting the set on ice, I use 300mA for 1.5 hr to transfer the proteins to the membrane. It is related to the size of the protein, for smaller protein you need a shorter time, and for larger protein, you need more time to transfer.
I agree with Jeffery, low volts, cool, and longer time for transfer.
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