Dear Scientists
I ran my SDS-PAGE with 37.5 microliter protein + 12.5 microliter sample solvent in every well and with stacking gel with pH=6.8 and separating gel with pH=8.8 with 100 voltage for 2 hours and now this is my gel. Can anyone help me figure out what the problem is?
- The lane in the middle is overloaded, use less protein.
- There is no front marker, you probably ran the gel for too long. Stop the electrophoresis when the marker dye has reached the bottom of the gel, but has not yet left it. Otherwise, you can't calculate Rf-values.
- In the bottom quarter there is a band going through the entire gel. If this band corresponds to about 60 kDa (you did give any scale), then it is keratin from your skin. Use gloves when handling anything used for PAGE, including the sample buffer.
- The bands seem wider than the original wells. This can happen if the wells contained different volumes of sample (use water/buffer mixture to make sure all wells - even those w/o sample - contain the same volume), or too much salt in the samples (if need be, precipitate proteins with MeOH/CHCl3).
- The bands appear slightly fuzzy. Make sure that all your buffers correspond to Laemli's recipe to ensure proper stacking.
- Try to keep the stacking gel until after staining. Sometimes proteins do not enter the stacking gel or move only to the interface between stacking and separating gel. If such problems occur, at least you want to see them.
- Your dye seems to have precipitated, leaving speckles on the gel. Filter before reuse.
- One lane should contain marker proteins. Personally, I prefer pre-stained markers where different proteins have different colours, like Rainbow-Markers.
- Make sure that all equipment is scrupulously clean. Dye residues can be removed with a little MeOH.
For me it seems that some of your buffers is wrong. Check the pH of gels, as well the running buffer. In our lab we sometimes need to adjust it to 8.3 , while in original protocol it is not required.
You should load a well with marker proteins and check the gel. If picture will be fine, then try to optimize protein amounts in samples.
Along with buffer pH you should also check the protein isolation method. I think your protein is degraded. then run the protein marker. You should also run the gel for shorter time.
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