My SDS-PAGE gels run on Bio-Rad Mini Protean equipment are taking much longer to run than I expect; about 4x the amount of time that online info and experienced friends say they should take. The resolution is adequate but not fantastic, and the excessive run times are annoying - can anyone spot a problem?
I cast the gels myself using 1mm spacer plates, so the gels are quite small: 7cm length (including wells and stacking gel) and approx 7mL volume between the plates. The gels contain 0.1% SDS and most of my recent runs have been 12.5% polyacrylamide. I usually cast the gels the day before the run and store them overnight in the fridge, sometimes I store them 2-4 days but no longer. My gel buffers (Tris) are old but the pH is correct. The acrylamide (29:1), APS and TEMED are fresh.
I set up my runs in a cold room (4C) with pre-chilled running buffer (1X TGS, prepared fresh). I always rinse the wells with running buffer. I monitor run with prestained protein standards (Bio-Rad precision plus). If I run at 130 volts, it takes approx 4-5 hours for the dye front to reach the bottom of the gel and the prestained standards to separate. I just tried running overnight at 20 volts, and after 15 hours the dye front is less than halfway down the gel. I remember that when I did this years ago, running at 120-140V would produce heat even in the cold room, and would run in 1-2 hours. Now, the buffer doesn't warm up at all, and it takes 4x this long.
So far I have tried the following and nothing changed:
- 2 different electrophoresis power supplies (both run agarose gels at expected rate)
- 2 different tanks/lids
- 3 different gel chamber assemblies (the part that has the wires and creates the inner buffer chamber with the gel).
- Double checked there are no leaks and that buffer covers the wells
- Run an entire gel with prestained standards only to eliminate sample-related issues
I would love any suggestions - I can't work out what I'm doing wrong!
Did you get any solution of this problem ..because in my case same thing is happening. Running time is more than 6 hour at 120v for resolving & 60v for staking.
Try running your gels with these settings:
250 V constant voltage
30 mA current (for each gel)
I use this configuration on Bio-RAD Mini Protean apparatus and it takes about 70 minutes to finish. If the problem persists, check and/or replace your buffer and try using a 2X solution.
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