VERY SLOOOOOW running gels (SDS-PAGE) - (Nov/01/2007 )
Hello,
this is a recurrent problem in my lab for over a year now I would say; in that time span I've definitely made up new buffers at least a few times and it always happens, not sure why.
The problem is that the gels seem to run extremely slowly. I'm running a 12% gel right now (SDS-PAGE, 3.5% stacking, 12% sep) and I'm over 1 hour into the run with easily another hour to go, if not longer to go. The sample/dye has just barely made it into the separating gel. I'm running at 100 volts (anything higher and the buffer will be too hot). 6% gels are no better really, the running time is still ridiculously slow. EDIT: Total run time (or rather, until I was sick of waiting) was 2 hrs and 40 min. I just thought of one other thing that might affect the run of the gel - I normally seal the bottom by pouring a small amount of agarose around the bottom end of the glass plates and allowing it to solidify before I pour the actual gel, but this results in the very bottom 2-4 mm of the actual gel being composed of agarose and not polyacrylamide....could this be the reason for slow running?
Could this mean I need a new supply of Tris or Acrylamide/Bis? I mean an entirely new bottle of the actual chemicals, not just remaking buffers; as I said, this is a problem I've been having in my lab for far too long.
Also I've noticed that the efficiency of my transfer buffer is ok if it's just newly prepared (I can transfer at 100 volts on a small powerbox we have, 1 hr is enough) but a couple of weeks later the same buffer and powerbox combination is unable to transfer at much more than about 50-60 volts (tops out the amperage on my powerbox; I tried another powerbox with a higher amperage and it was able to maintain the voltage at 100 but the buffer got extremely hot). This could indicate that my buffers are not keeping for very long even when stored at 4 degrees where we normally keep them.
One last question - could it be that my loading buffer is causing my samples to migrate extremely slowly? This is the only thing I can think of that I haven't made up in the last year, everything else has been made and remade at least a couple of times. The dye front is very blurry and not well defined at all; it collapses into a small/concentrated space only very late in the run and I've noticed that my smaller proteins and marker bands are very poorly resolved even when using 15% gels.
I'm very seriously considering moving to precast gels....
Thanks....very frustrating problem...
H
EDIT: Sorry for the very long post!
Have you ever tried an other powerbox?
I'm not sure it's due to agarose, but have you ever tried w/o agarose? could you use acrylamide with twice more temed and APS to polymerase faster, to fill the bottom of the gel instead of agarose?
are you pouring minigels? you should not need to seal the bottom with agarose. Check that your plates are not damaged or buy new plates and be very careful. clean, wipe and store them immediately.
Could you give us the recipie of your gel, so we could check if there is any mistake.
when I do 12% SDS-PAGE, 100V, It takes me 2 hours (for 6cm height). but I could increase the voltage, it will not be too hot. Give us your recipie of migration buffer. please give details.
may be too much salt in the samples, or too high ionic strength in the running buffer
the buffer shouldn't be getting hot (not at the voltage at which you are running, the gel yes, not the buffer).
check that you are making the correct concentration of the buffer components and that you are diluting it appropriately (if it is a concentrated stock (it should be)).
the buffer shouldn't be getting hot (not at the voltage at which you are running, the gel yes, not the buffer).
check that you are making the correct concentration of the buffer components and that you are diluting it appropriately (if it is a concentrated stock (it should be)).
thanks for the replies: here's the composition of the buffers I'm using:
4x 'running buffer' - 48.44 g TrisHCL, 230.40 g Glycine, pH- 8.2-8.3, volume to 4 L, stored at 4 degrees. Our 1x running buffer contains 0.1% SDS final concentration.
Transfer buffer is made up of 1:1:3 ratio of 4x running buffer:methanol:water (i.e. in 1L there is 200 mL of 4X buffer, 200 mL of methanol, and 600 mL of water).
I'm currently running precast gels with the buffers from the company who makes them and I calculated from the composition of their buffer that they are using 2.9 g of Tris and 14.4 g of Glycine, 10 g SDS in 1L of buffer whereas the buffer we've been using for a long time in our lab contains 12.11 g Tris, 57.6 g Glycine, and 10 g SDS per 1L.
This precast gel is running far better than any of my own gels and I'm running it at 150V (as per manufacturer's instructions) compared to my 100 V.
so are my buffers just entirely wrong?
H
your buffer may be too strong. this makes it more conductive, higher current at lower voltage leads to greater wattage (more heat). the higher concentration buffer makes the sample run slower.
are you sure your buffer is a 4x?
I sealing the bottom of the gel with an autoclave band instead of pouring agarose to the bottom of it eliminates the agarose parameter. Are you tried to run your gels in a cold room to decrease the temperature when you applied more than 100V? Do you ever change your procedure totally? I mean try a different protocol. I am sending a differnt protocol to your PM. If you have chance i thnk you should try.
are you sure your buffer is a 4x?
That's what my protocol says - "4X running buffer".....so yeah, at least according to this its 4X.
I'll check my PM, thanks for your help.
I suppose it could just be the agarose, but I have a suspicion that my sample buffer may be junk: I recently ran a precast gel using buffers made for this gel by the company who sells them and my samples still ran a little funny - running it again using their sample buffer to compare results.
Thanks to everyone who replied.
H