3 replies to this topic

[JesseC]

Hi everyone, I'm new here and am just starting in a lab and have run into a problem that is driving me up the wall.  We are trying to look at proteins around 14 to 16 kD and for some reason our gels look very squiggly and ugly around that size.  Higher molecular weight bands look fine, the gel only begins to look that ugly around maybe 20 kD or so.  The dye front does not run very pretty though and begins to give that squiggly appearance when it is about halfway through the gel.  It has been very difficult to discern anything about our POI because of how ugly the gel is and I would really love any advice that could help me out here.  We're using a mini-protean tetra, and using pre-cast gels, 4-20%.  The gels are about a year old, but they aren't expired or anything.  We're using RIPA to lyse, a pretty standard recipe of it that I've found many places on the web.  After a BCA assay, I add Laemmli with 15% BME, boil at 100C for 10 min, load whatever amount I need to get 20 mg of protein from each sample onto the gel, and then run 120V for 1 hr.  We've also tried slowing it down to 60V for 2 hrs, with minimal difference.  We've made and remade the NaCl we're using in the RIPA to make sure its not a salt problem, and that hasn't made a difference either.  Any ideas here would be greatly appreciated, thanks!

[mdfenko]

it's not so much the quality but the quantity of the salt that causes problems in page.

problems such as the ones you describe are usually caused by the buffer contributed by the sample. you can prove this by dialyzing away the ripa components (you can perform drop dialysis on a few microliters) and running that on the gel.

[JesseC]

Thanks for the response mdfenko, I appreciate it.  We are using a 6x sample buffer, so I wonder if using a 2x buffer would dilute the salt out from the RIPA a bit more and help with this issue?

[mdfenko]

it won't really help, you'll have the same mass of salt present. you can drop dialyze your samples to remove salt.