I'm currently working on the optimization of the trypsin digestion protocol (duration and trypsin concentration) of peanut extracts (with Tris and Urea). I use SDS PAGE (12% BioRad) to check the completion of the digestion.
I use 2 different coloration techniques : coomassie blue and silver staining. The silver staining gives strange results as you can see. It seems that the bands are colored everywhere, excepted where proteins are located (clearly visible on the 5th band with a non digested extract).
The coloration technique seems to be good since the 6th band, which is only a control with trypsin alone, works normally (no brown coloration, only the spot at 25 kDa).
Has anyone experienced that at all? Could it be due to lipids which are very abundant in peanuts ?
In my experience, lipids run at the bottom of the gel at the front, but can extend upwards from there if abundant enough, causing distortion of the bands. This does not look like that situation.
I don't know what is causing the inverted staining, but it seems to me that Coomassie staining will be sufficiently sensitive for your project.
Dear Maxime:
Are you working with redox proteins? Some redox centres produce this effect on silver stain. Consequently, this method is not the best to stain gels, when working with metalloenzymes containing redox centres at high concentration (Fe-S for instance is one of them).
Best wishes,
Rosa María
Dear Maxime:
I completly agree with Pavel Majek - try lower amounts of Protein / lane at silver stain. I also think that the inverted stain is an artefact induced by "overloading/overstaining" the gel.
Kind regards
Wilhelm
Dear all,
Thank you very much for your answers. I'm gonna try some extra dilutions of my sample.
I also find that peanut are rich in aldehyde and ketone, reagent use to reduce silver in this type of coloration. Maybe is it also a part of the problem.
More experiments soon !
Maxime
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