Based on your picture, I suppose the problem is in loaded amount of protein - so try to load less or dilute the sample...but it all depends on purpose of the gel.

Based on your picture, I suppose the problem is in loaded amount of protein - so try to load less or dilute the sample...but it all depends on purpose of the gel.

U can dilute the sample, check the concentration of gel and EDTA used

Try to use 100-500ng/Lane protein WB.

Dear Eiman, I see two problems: too much of protein introduced into the lanes of the gel, and too long incubation with antibodies. In addition, reduce the time of membrane staining.

Dear Eiman: if you only need 3 lanes, you can load 1/5th of what you have loaded and another 3 lanes loading 1/10th.  A faint band on a coomassie-stained gel can indicate as low as 0.25 micrograms of protein, in your gel I can see more than 10 times that so you can definitely load less. Also, destaining 2 times on 10% acetic acid (for 20') can improve your destining an you'll get clearer bands when you load less protein. I you couldn't destain enough leave it overnight in water or 2% Acetic Acid.

Cheers!

Thank you all for your answers. I tried another protocol to extract sperm membrane protein and it's seems it works. I think the problem is using this ready kit to extract DNA, RNA & protien, so maybe a contamination occurred during the process.

I think you should stick to your previous kit for extraction with diluted sample. Then of course try another kit if getting same types of bands.

The concentration of the studied protein in your sample is high. It is better to dilute the sample or load lower amount.

Given that you have time, you could run a gel with various protein concentrations, e.g. you've got 7 empty lanes in the above pic. You'll be optimizing what protein concentration it's the most beautiful for your publication.  

Also, if this is too much, I would reduce the staining time. 

Regards from SF

i also suggest try to load less amount of sample.

The thick band is possibly due to albumin contamination.

Try washing the sperm in serum free HBSS or PBS.

I totally agree with Mohammed´s comment. Likely albumin contamination. Good Luck!