You potentially overloaded the gel with excess protein and assuming your protein is the larger one, it has a lower molecular weight impurity which you can remove with gel filtration in a superdex G-200 gel filtration resin.

If you put your gel in water in a plastic container in the microwave for about 1 minute you can easily destain the gel so it doesn't look so blue.

Too much salt in the sample?

I am also doing the same experiment for protein and facing the same trouble if someone knows it is my question.

Perhaps try using a gradient gel for better separation if you have not tried already. 5-20% or 3-18% and allow the ladder to run down to the gel until the bands separate well. Also, consider doing a titration of your protein to see the optimal concentration for loading. Is your protein purified?

Hi Evi and Hasnat,

Firstly , please mentioned/mark your protein size. You can use Bio-Rad SDS gel recipe. Use fresh SDS running buffer .Check the pH of Separating and stacking buffer, It should be 8.8 and 6.8 respectively.

1st, mentioned the protein size ;2nd, mentioned the gel concentration;3rd，select a appropriate electrophoresis condition.

Hi Evi and Hasnat,

First, check the size of the protein of your interest, then choose the % of gel as per it.

Second, check the concentration and the pH of each reagent you are using for the experiment.

Third, check the volume and concentration of your loading dye.

Fourth, try pre-running the gel prior to loading the samples for at least 3 minutes.

Note :- while isolating protein during the downstream processing try to pipette out the protein without getting any pellet/cell debris contamination.

Hope you find it helpful.

Dear Evi Giouroukou

1-what is your method for protein extraction?

2-Do you use fresh Glycine Buffer 1X?

3-is your coomassie blue buffer fresh? (in a long time methanol may be loose)

4- Don't forget to get a beautiful band, use 2x loading dye in free wells.