What is your source of protein?

It can be form dimer, aggregate or etc?

Dear narges Jamal,

Please let me know your source of protein sample.. also lable the protein marker band sizes.. and re upload the gel..

If possible get any commercial protein of 20kda to run control experiments or you can run sds with egg protein samples along with your marker.. then you can come to an conclusion that.. weather your sample doesn't contain the required size protein or your experiment having some problem..

If protein is there in your sample mostly it won't disappear.. might be the concentration of your protein is low.. try to purify the required protein if possible and concentrate the same.. then run sds...

You can do the silver staining too for such kind of problems...

Hope this information is useful..

All the very best for your research..

Amended on 22 October 2018

I must sadly and repeatedly declare that polyacrylamide gel (PAG) is extremely strong hydrophobic material. Hydrophobic membrane proteins can not be electrophoresed by notorious SDS-PAGE due to absorption onto the gel (please see file; IEF for hydrophobic protein).

Further, I have found that connectin/titin (the main protein of adult liver cells; please see files "HepG2 Fucoidan" and "JMBT alopecia") can not be entered into both 12 and 15% gel, which has been appeared on the top of your gel. Connectin/titin may also be interfering to elute another small proteins into the gel. Connectin/titin is present in adult liver cells and adult serum at median Mr 2,070,000 (range; 790,000 - 3,390,000; adult specimens n = 6).

High Mr proteins seem to be strongly bound to PAG gel, but small Mr proteins must be fixed by acid due to weaker van der Waals' force.

Prevention of van der Waals' force is most important issue in RP-HPLC (please see file; Lysozyme by RP-HPLC). I am estimating that Mr less than 100,000 can now be safely eluted from the ODS column. Therefore, cutting of large proteins by protease may be required for RP-HPLC purification. Therefore, lipoamidse (Mr 140,000) purification is performed in low yield (please see file; Purify Brain Lipoamidase).

Naresh Kumar M Alireza Mordadi Kou Hayakawa

Thank you for your valuable comments. I discord the problem yesterday , and that was "Fixation" .

The protein bands all appeared when I fix the SDS-gel in methanol- acetic acid solution prior to staining by coomassie blue.

Instead of fixing and staining..

You can prepare staining solution with 10% acetic acid , 50% methanol and 40% water along with 0.25% CBB-R250.. this solution will work well...

For destaining

10% acetic acid, 50% methanol and 40% water..

This will work well ..