I would like to check protein profiling of plant protein through SDS PAGE but whenever i run SDS got two unwanted lines within the gel which hampered protein running on gel.. please anyone give me suggestion to resolve this...
for your observation a picture of the gel is attached here with.. first line is of marker...
thanks
Hi,
Something problematic with your dye or SDS buffer. Please cross check Dye and buffer preparation manual.
Rajkumar
Thanks for your suggestion.. I have used fresh loading dye for the SDS and i have repeated the experiment 2-3 times but got same problem.. even first two and last two well are empty but still lines appeared in that...
I suggested that running buffer to containing tris-glycine proper percentage per volume.
I think this is the problem with yours commassive staining dye. Prepare fresh staining dye and/or stain your gel for less time. hope it will work.
check the running buffer pH as well as the separating gel pH and use loading dye
6X LOADING DYE 10 ml
Glycerol 4.7 ml
Tris-HCl, pH 6.8 1.2 ml
DTT 0.93 g
SDS 1.2 g
Bromophenol blue 6 mg
Distilled water 2.1 ml
I also think that is because of loading dye and I believe it should not hamper your other proteins. I would suggest run gel untill those two band come out of the gel or atleast in the bottom of gel. Later stain and de stain as usual and you might get rid of those two bands and gel the resolved bands of your protein extract. Why do you stop you gel in half way? let it run fully once. sometimes dye diffuse and might appear like that.
You could run only the buffer to understand if the problem is linked to a reaction between plant proteins and dye, or if it's a technical problem....I think that protein samples contain a substance that reactives with sample buffer and create this problem. You could change something in extraction procedure.
i would suggest you to make fresh reagents and running buffer, pH should be proper, check your dye and if you store the chemicals in any type of plastic bottles that might be the cause due to formation of mold proteins.
http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gelgoofs/bizarre.html#4
These two unwanted lines can be cuased by reducing agent you use. The false and obstructive lines are usualy appeared if 2-mercaptoethanol is used in loading buffer or extraction buffer, it is known occasion. The other reagen, DTT, can als give such false band if used in concentration higher then 100 mM and th pH of separating gel buffer is not appropriate (seems to be more acidic).
I support Gopal Tiwar,
Because, may be your protein degrades into peptides and moves along with dye front show two bands at the end?????????????
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