Protein ladder not separating - (Mar/10/2013 )
I have run several SDS-PAGE with 10% resolving and 5% stacking. I always couldn't resolve 25kb protein ladder. After 50kb marker, the rest seems to stack and not separating. There is one time, the dye front even stop early even though the current is running.
Is that a problem with my gel or buffers?
Thanks for help!
Most protein dyes are pH sensitive. Did you loading dye or ladder change color as it migrated down the gel? Are you using pre-made gels or are you preparing the gels yourself? If you are using pre-made, are the gels expired, have they been stored properly? If you are making the gels yourself, it is possibly that one of your buffers is contaminated or the pH is off. Have you made any modifications to your western blot running buffer? Are you using a new stock of a chemical (i.e. SDS, Tris, Glycine, etc.).
Is anyone in the lab having similar issues with that particular western blot rig? Are you sure your apparatus is maintaining the correct voltage? What is the temperature of your running buffer in your western blot rig (warm, scalding hot)?
Think about some of these questions and it will jump start your troubleshooting.
Thanks! the loading dye didn't change color. I am preparing the gel myself. There is one lab mate having the same issue. I was thinking it might be the issue of running buffer since the only thing we use in common is the running buffer. Running buffer is usually 700ml H2O, 200ml 10X running buffer and 100ml methanol. The voltage I typically use is 60V for 30mins, and 120V for 1.5 hours until the dye front reach almost the end.
Because of the stacking of protein ladder, sometimes my dye front stopped early.
Yeah, just try remaking your buffers and see what happens. You add methanol to your running buffer?
methanol in the running buffer will strip sds from the protein and will cause migration problems.
we have seen tracking dye stop early in the run. we found that if we continued the run that the dye would "jump" to the bottom of the gel, sharpen and continue to migrate. the resulting stained gel appeared normal. we think that it was caused by aging of a component in the running buffer (most likely the sds).
if you need to see separation of a wide range of protein sizes then you may want to consider running a gradient gel.
the information that comes with the size standards (or can be found at the manufacturer's website) should show you how the standards should appear when run at various gel concentrations.
Erm, so your running buffer is at 2x concentration? You should only need to add 100ml 10X buffer to a 1L solution.
And I haven't heard of adding methanol to the running buffer, to the transfer buffer, sure, that is common, but not to the running buffer. Have you mixed up your protocols perhaps?
My bad, no methanol in running buffer. Just 1x running buffer. So, I have figured that out, the protein ladder not separating because of the leaking problem in the cassett.That's what I found in troubleshooting for a week with new buffers. Thanks all!