SDS-PAGE running strangely - (Jun/17/2004 )
when i run my SDS-PAGE, it seems that themarkers don't fully separate out [there are meant to be 7 marker bands spread evenly, but only 4 appear, spread out with wide gapd between]...... does this mean that the smaller bands are not separating out? is this due to running conditions [200V for 1 hour)
It depends on the acrylamide concentration. But genaraly the small proteins separate first. Your problem could be that the smallest proteins run out from the gel. Maybe you should run the electrophoresis shorter.
Could be worth getting hold of a sample of colour markers as well to check the progress of the gel. Could also be that the markers you have need boiling and reducing to behave as expected.
is it possible that proteins will run ahead of the loading dye? if so then maybe the samples are running off the end of the gel
The gel is deficient in cross-linking.
this most probably seems to be the case.
We were having the same problem in our lab - bands running faster than the front line, very bad resolution, missing bands, additional nonexistent bands etc...
Never really figured out the real cause but the solution was simple: to replace buffers used for sds-page..
Namely, the running buffer, sample loading buffer, buffer for resolving gel, and bufer for stacking gel. A common problem is pH: These buffers are concentrated tris solutions. Such solutions are known to give wrong pH when cheap pH electrodes are used. Therefore, ALWAYS check your pH with strips - or just don't use pH electrode in this case at all.
The problem could be that very low percentage gel has been used.or else there could be coalescence of protein in marker which usually can be separated by boiling in loading buffer for 7-10 min.
For the marker to produce the ideal ladder, follow the manufacturer's instructions carefully. Some markers require boiling, while others do not. Some require a minute long incubation at 40C to dissolve precipitates.
Most important things, check the percentage of the gel the manufacturer used to produce the ladder. Generally 12% gel is used. I had mistakenly used 8% gel and some of the bands didn't separate out well.
Check for expiry of the marker. I once used an old Sigma marker and many of the bands were missing.
Is your marker a premixed soultion or separate powders which you reconstitue and pool. In the latter case, be sure that all the standards are mixed in equal molar concentration ; otherwise bands of unequal intensity ( probably missing) could result.
If you are employing silver staining then check out the manufacturer's instruction for the best staining procedure.
For best resolution you need to run the gel completely; that is, till the blue front reaches almost the bottom of the gel. Some of the bands separate out only after half the run has completed. You will notice this if you use prestained markers ( which are visible during the run itself).
Just an offhand suggestion: try running at a lower voltage ;say 80-100V
Could anybody suggest the reason for this case: my protein actually 30kDa but on SDS PAGE it migrate at 37kDa?
Is that the protein surface charge that involve in the migration of protein on SDS PAGE?
Thank you in advance.