The samples that you have run are cell lysates I guess? In such a case, cell lysates contain many cellular proteins besides your desired protein. Probe sonication is way more efficient in cellular lysis as compared to bath sonication which explains your more intense bands in the probe sonicated samples.

Tushar Chakraborty Sir, Thank you for your valuable answer. In both the sonicated samples I have removed most of the cell debris using a 0.45-micrometer membrane filter. Then, how can I confirm my desired protein? I don't know the exact molecular weight of my protein. In this case, after the literature survey, I came to the conclusion that the protein ranges from 17kDa to 30kDa, it differs even in the same species based on the strain. How can I confirm my desired protein?

For detection of low molecular mass proteins, a tricine-system (doi:10.1016/0003-2697(87)90587-2, 10.1038/nprot.2006.4) is better than standard SDS-PAGE.

If you want to detect a specific protein, then you need a specific reagent, unlike CBB, which stains all proteins. You could for example use an antibody after blotting your gel. Another way is to detect its enzymatic activity (zymogram), but that is more commonly performed after non-denaturing electrophoresis.

A more pragmatic approach is to repeatedly perform electrophoresis during purification (using a different assay to identify the appropriate fraction), the protein of interest is the one whose band intensity increases ;-)

Your gel doesn't look too bad actually, it just could take some additional destaining.

Engelbert Buxbaum Thank you for your valuable answer and compliment about the gel sir. Am happy with your reply. I will check the reference that you have mentioned above.

Hi Srichan., the previous responses are good ones however, I wish to add.

Your SDS-Gel looks like you have uninduced and induced proteins ran on the gel that's why you have faint and thick.

Regarding your protein weight, while your protein ladder should serve as a guide, look at the picture I attached which should likely give you an idea of the desired band.

I hope this helps?

Please note the duration of the procedure and the voltage.

A proper voltage improves the speed at which proteins(samples) move and how they bind.

Raphael Galleh Sir thank you for your response.

Roya Mirzaei Thank you for your response mam. the duration for the procedure was 1.30 hrs and the voltage was 80 and 120.

Crude lysates tend to look like that, even when filtered through a 0.45 um filter, if you already have a range of proteins and your protein doesnt have a tag to isolate it; i could suggest molecular exclusion filters (amicon, Merck); a 50 kDa filter will remove a bunch of those unwanted proteins and concentrate the ones in weight range (not all though); have you confirmed your proteins by WB?

As you mentioned earlier, you suspect the molecular weight of your protein to be between 17-30kDa. Do you know your desired protein (protein name/sequence/gene ID) ? Because within this molecular weight range, there will be multiple proteins. As you don't know your protein, neither is there a way to clone & then induce your desired protein expression nor perform a western blot.

If you have the option, perform a LC-MS to confirm your protein of interest.

Carlos Alberto Gonzalez-Villarreal No sir, I haven't done western blot yet.

Tushar Chakraborty Yes sir. Its pili protein.

Use fresh prepared acrylamide-bis-acrylamide solution from fresh stock. Powder form shouldn't be very old as it is giving fuzzy appearance instead of crystal clear after gel casting. And even check for APS and TEMED. APS freshly prepared and use.

If you doing protein expression in Bacterial cells like BL21 or Rosetta, after 20-25 min of IPTG induction, add 200 microgram/ml of Rifampicin to induced culture and further incubate in shaker. Addition of rifampicin removes background proteins transcribed under bacterial promoters.

use freshly prepared protein samples , you can load less amount (50 -70 μL ) protein sample in each well, after the CBB staining destain the gel properly or directly you can go for the silver staining of gel.

Prepare fresh solutions of APS and SDS for the gel preparation. And you can try silver staining too.