Analysis of common strip problems in electrophoresis experiments

First, the smile zone (both sides are raised, the middle is concave):

Causes of formation: mainly caused by uneven solidification in the middle part of the gel, mostly in thicker gels.

Treatment: Allow it to fully solidify before experimenting.

Second, frown-shaped zone (both sides down, middle bulging):

The reason for the formation: mainly occurs in the vertical electrophoresis tank of the protein, which is generally caused by the fact that the bubble at the bottom gap between the two plates is not excluded.

Treatment: Add an appropriate amount of buffer between the two plates to remove air bubbles.

Third, the zone trailing:

Causes of formation: mainly caused by poor dissolution of the sample or excessive concentration of the separation gel.

Treatment: Centrifugation before sample addition; select appropriate sample buffer, add appropriate amount of sample to promote solvent; run time buffer for too long, reconstitute; reduce gel concentration.

Fourth, the zone appears texture:

Cause of formation: mainly caused by sample insoluble particles.

Treatment: Centrifugation before adding the sample, add appropriate amount of sample to promote solvent.

Five, ghost belt:

The reason for the formation is mainly due to the fact that the reducing agent is oxidized and loses its activity during heating, so that the originally dissociated protein molecules are refolded and recombined with the subunits, and polymerized into macromolecules, the molecular weight of which is larger than the target zone. Sometimes it is not possible to enter the separation gel. However, it has the same immunological activity as the target region, and can be seen in the immunoblotting reaction, and can act as an antibody corresponding to the target region.

Treatment: After heating and boiling, add appropriate amount of DTT or Beta mercaptoethanol to supplement the insufficient reducing agent; add appropriate amount of EDTA to prevent oxidation of the reducing agent.

6. Bromophenol blue does not serve as an indicator:

Reasons for formation: In the experiment, it is often encountered that bromophenol blue has run out of the bottom of the plate, but the phenomenon that the protein has not run down is mainly related to the concentration of buffer and separation gel.

Solution: Replace the Buffer with the correct pH value and reduce the concentration of the separation gel.

Seven, the zone is very thick:

Cause of formation: mainly because the concentrated glue is not concentrated.

Treatment: Appropriately increase the length of the concentrated gel to ensure that the pH of the concentrated gel stock is correct (6.8), and reduce the voltage appropriately.

Eight, the electrophoresis voltage is very high and the current is very low:

Cause of formation: Mainly because the electrophoresis tank is not properly assembled, the current does not form a passage.

Solution: The electrophoresis tank is assembled correctly.