how to get a good band on SDS PAGE analysis for spirulina protein. In my results showed dye staining bands on the sample loaded well. The same procedure was follow on BSA sample given clear band on appropriate Mw. In the same way 10% to 15% gel concentration was follow but showed same result dye stained gel with protein band ?
Hi there,
protein sample doesn't even seem to enter separating gel as you get coloration at the very top of it. You are certainly confronted to protein precipitation/aggregation during sample preparation. You may use urea to improve solubilisation and more reducing agent as well as extend temperature treatment prior loading onto gel.
Is the left lane the one with BSA? If yes the pattern is not very neat (diffuse band and smear) and I suggest to consider to improve gel preparation.
Actually, some proteins (especially hydrophobic) prefer rather lower temperature for longer time with the loading buffer.
Did you check the protein concentration? I think you should use different concentrations of your test protein and resolve on gel. Hope it will help for you.
In my view i think there isn't enough expression of protein. The faint bands observed could be as a result of low protein concentration.
I will suggest that you increase the hours of expression after induction of protein and also increase the duration of lysis of the cell for release of enough protein.
Consider increasing duration of heating and amount of denaturing agent added to the protein sample. This step will enhance band separation in the resolving gel.
All the best.
You might consider the following:
Diffuse bands; possible degradation too (ice usage while loading)
Are u storing on ice post denaturation?
Protein concentration looks low too ( reason for invisible low abundant proteins)
U have resolution issue also here. Pls follow the Sambrook protocols strictly till u get sharper gels with good resolution
I had the same problem for my protein. I think these are because of high temperature. Change the running buffer and have new stocks for preparing gels.
It could be due to an incompatibility between your sample buffer an the loading buffer: high salt concentration (high phosphate concentration) or pH incompatibility in your sample could cause precipitation or retardation on the gel.
I think you should add maker or sample for control, running very slowly at 4oC.
PLEASE CONSIDER FOLLOWINGS;
1. Firstly you have to quantify the concentration of your protein
2. Tris buffer pH kindly check also the tris glycine buffer
3. Loading dye fresh prepare
4. Acrylamide and bisacrylamide % salution
I've only ever seen results like this on proteins that had degraded.
I shoul say that your protein is degradated and probably contaminated. Are you using gloves and really clean equipments. Trypsin from your hands could be present in your sample too.
Thanks for all to gave valuable suggestion. Now i successfully finished my electrophoresis step. I understood my mistake, presence of high pigment content in my sample so i got that result. When i removed, get a clear bands in their appropriate molecular weight.
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