2-D electrophoresis problem... strange spots - (Apr/19/2006 )

Dear all,

I've got some problem with my 2-D. Since i've been encountering horizontal streaking problem, i've used amersham 2-D clean-up kit to remove the impurities from the sample. In my gel (self-cast 10% separating acrylamide gel with stacking), while there are some distinct circular spots, some of the prominent spots appears as an extended circle vertically, just like a water droplet. I think there should be some problem with the second dimension. But i've tried to run several gels at the one time with my labmates who're using different samples, and she didn't got this kind of problems, so i wonder if it's really a second dimension problem. In addition, some spots appears as orange color after staining, differed from the expected brown spots (i'm using silver staining), and they tends to be larger spots. What could be the reason?


Thanks for reading~

i have seen nucleic acids (specifically, trna) stain orange with silver. we used trna to aid in precipitation of low concentration protein prior to loading on a gel. it doesn't stain with coomassie but when we started to silver stain we saw large orange blotches in the gel.

hello,

some of the "tear drop" shaped spots might be results from precipitation that occurs in the IEF separation. if the proteins precipitate at their pI, only some of that may be re-solubilizing when you equilibrate into your SDS-PAGE buffer and run it out. the remaining insoluble protein may partially re-solubilize over the course of your SDS-PAGE, causing the water droplet effect you see. another possiblity is that your self-cast gel contains keratin contamination (or your loading buffer). this often leads to artifacts that are called (creatively) keratin tears. you could run a blank gel w/ buffers included, stain it, and see if that's the case. as for your orange spots on a silver stain, not all proteins stain equally- w/ any stain for that matter. metalloproteins often bind the silver, which results in orange-ish stains, if not semi-clear stains.