I have been doing a western blot with the transfer condition of 350 mA (about 100 V) for 1 hour 10 mins at 4 degree. The transfer works fine when I am doing only one transfer at a time. I wanted to know whether the transfer condition will vary if I do two transfers at a time i.e. transferring proteins from two gels to the membrane in the same apparatus. Thank you.
Dear Ishan
- I have never personally seen a difference between transfer for particular conditions for one or 2 gels
- If you have any concerns, particularly if your protein is larger (>50kD) then it is not uncommon for persons to transfer for up to 3 hours; You could therefore try transferring @ 4C using the current and voltages you describe
Hello,
I have been transferring two gels at a time for Western analysis without any issue. You can transfer for 3 h, at 100 V, at 4 degrees ( I have been doing the same). If you have any issues in transferring i suggest you to increase no. of scotch pads or no. of filter pads. hope this helps.
Will work fine, as the others have said. My only suggestion would be to add a magnetic stir bar (a mini one) and have it on low-medium spin speed to maintain even ion distribution and temperature gradient - might ensure more equal tank conditions for both transfers. Good luck!
I do not expect any changes in transfer conditions. i normally run mine one at a time (30V for 90 minutes at 4C), but I have done two a time several times. However, I would suggest adding extra sponges in between the two sandwiches in order to prevent protein transfer onto the other membrane.
If you're planning on quantitating your signals on your blots afterward (e.g. by doing densitometry), make sure you use double membranes and probe the back one as well to control for blow-through. Also, make sure you run each sample at two different concentrations (i.e. 1 unit and 5 units), so you can show that your quantification is detecting these differences, and that your assay is therefore within the dynamic range of your membrane's ability to bind protein, your antibody dilutions aren't limiting, etc.
Hi, I was transferring 2 most of the time and had no problem but once I got the same problem as you have mentioned. The magnet and @4C is definitely the best option. since I never had any problem when I was using the same method. It happened when I did not have access to 4C and I did the transfer covering the container with ice and using no magnet. For me transferring 1h30 min works perfectly. I am working with the range of 40-180 kD proteins. Wish you luck.
The resistance may be different in two conditions.First case is less in one gel condition compared to two gel condition. It means there should be some more time needed for the proper transfer in both gels. eg: If you setting 350mA it will show around 100v in one gel condition but in the two gel condition that might be less than 100v it means whatever time you giving for transfer is not enough for proper transfer.So accordingly you can adjust the timing and i hope you will get good transfer.
Best of luck
Thank you all for suggestions. It worked fine without changing any condition.
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