Urea

Poor quality of reagents

Use only the highest quality reagents

Issues related to Urea

Only use freshly deionized urea.

Low molecular weight species have diffused

Begin electrophoresis as soon as the sample is loaded to prevent low molecular weight species from diffusing.

Poor stacking

1. Only use gels that were recently prepared

2. Add a stacking gel or increase height of the stacking gel. Prepare the resolving-gel surface by first rinsing it with stacking gel monomer before pouring the stacking gel to ensure continuity between the gels.

3. Check pH values of the separating and stacking gel solutions. Do not backtitrate buffers.

Incomplete gel polymerization

Allow the gel to polymerize fully

Running conditions

Conduct the separation at a lower current or voltage setting

Too much TEMED or APS (Ammonium persulphate)

Lower the amount of TEMED or APS

Sample incorrect prepared

Sample preparation:

a. Dialyze or desalt the sample.

b. Centrifuge or filter sample before loading to remove particulates

c. Reduce the sample volume or concentration.

d. Improve dissociation of protein subunits by heating sample in SDS (Sodium dodecyl sulfate) sample buffer 1-2 minutes at 100 °C. Store on ice after heating.

e. Store sample on ice before it is denatured.

f. Add protease inhibitors such as PMSF (Phenylmethyl-sulfonyl fluoride) if necessary to prevent proteolytic degradation of sample.

g. Add more mercaptoethanol or dithiothreitol; check sample treatment.

h. Make fresh SDS sample buffer

i. Use the same buffer for the sample as for the stacking gel.

j. Increase the amount of glycerol or sucrose to increase sample density.

k.Store samples to be frozen in aliquots to prevent repeated freezing and thawing.
Store at -40 °C to -80 °C.

l. In a continuous buffer system, the sample may be too dilute. Use a discontinuous buffer system with a stacking gel, or a more concentrated sample.