I made two 10% sds-page gel (10% resolving and 4% stacking) and run them at 20mA. I am testing whether my newly edited recipe for gel casting is correct or not. Apparently, the gels took 3hr for the dye front to reach the bottom of the gels. The problem is somehow the protein ladders started to separate within the stacking gel, there are 4 to 5 bands above the interface and the remaining bands at the resolving gel.
May I ask anybody who knows how to resolve this issue or not?
Have you freshly prepared the reagents for polyacrylamide preparation?... (TEMED, APS, acrylamide solutions)...Your gel polymerization seems non-homogenous according to the given information...If the resolution starts at stacking gel, the experimental percentage is probably higher than the theoretical gel percentage which may have originated from improper application or the aged reagents (inaccurate polymerization)...
You indicated the newly edited recipe, what was changed, and can you able to verify without editing that the old recipe works well with the same reagents?
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