If you do a Bradford Assay with a protein standard under some range of protein concentrations, you will get a curve with a certain slope and intercept. If you do a lowry assay of a protein standard you will get a curve with a different slope and different intercept. Your actual protein concentration is probably somewhere between those curves. I'm not sure which concentration is actually real but they are reproducible and that is good.

So if you protein concentration is less than what you predict it should be, you could see if your protein is really there by doing a silver stain. A Coomassie stain can detect microgram levels of protein. A silver stain can detect nanogram quantities of protein. If your protein isn't there by silver stain then your protein isn't there.

What I know from doing silver staining in the past is to accurate measure silver nitrate. If your balance is miscalibrated you will overstain the gel, and I used some silver stain which used citric acid and formaldehyde. (There are so many formulations of silver stain but I trust these ingredients, and I don't exactly have the protocol when I did the staining procedure in another lab.) Once the bands start to appear add the destaining solution. Timing is important in silver staining, and it takes about an hour if you not using a terribly complicated protocol.

Make sure you are using the correct stain. There are 2 Coomassie Blue dyes.

SDS-PAGE strength seems fine, please make sure the proteins are not denatured,

Dear Yash Sharma would you please tell me how you prepared (25 micro gram) of your protein before loading in gel . and the amount of coomassie blue dye used. This could explain many reasons of why protein bands didn’t appear.

Dear Yash Sharma,

there might be several reasons for the missing bands, e.g.:

- too low protein amount (but 25ug should be fine)

- loss of sample (protein) during loading into the wells

- error with the electrophoresis

- error with the staining

If there is no staining signal in the whole gel (also in the wells) and you are sure that the electrophoresis went well (bromphenol blue line reached the end of the gel) then it is probably the last one. Please check your staining protocol again and run next time also other protein extracts (e.g. MW marker) as controls in parallel to verify if the staining or the sample is the reason for the missing bands.

Good luck.

Best regards,

Steffen

Dear Steffen

I have also run other samples on the gel to check whether there is any error, but i found the bands of my others samples on the gel.

Dear Yash,

ok, then something is wrong with your sample. You might check the protein determination again. If you haven`t done it earlier you should use the sample buffer without protein as control to see how much the components in the buffer already change the OD and in turn affect your final calculation. Otherwise Bradford might indicate a higher protein amount than you really have in your sample.

Have you used the same sample buffer and sample preparation as for the other samples? Have you heated the sample before loading? If all these steps are ok you could then load increasing amounts of your sample onto the same gel (e.g. 25ug, 50ug, 100ug) and check if you see some bands with higher protein amounts. Good luck.

Best regards,

Steffen

Hi Yash,

1) Your protein might have precipitated due to acidic pH.

2) Your protein concentration might not be accurate, you can cross check it again, I would advise you to check with Bradford as well as BCA.

3) You can run a small conc. say 50 micrograms of BSA as a control.

4) Also check with your staining and destaining reagents and with the time points. It can be possible that you had faint bands and go off with over destaining.

I also had protein purification, in my case acetone precipitation gave purified protein by there was no enzyme activity. So, you may try other methods of protein purification as well.

Hope my suggestions could be helpful in your troubleshoot.

Hi Yash,

in some rare instances it's possible to elute your protein from the gel upon washing, staining and/or destaining. This is particularly true for very small proteins, with a MW well below 10 kDa. You might want to try to fix your proteins to your gel before any subsequent steps, especially if your staining solution is water-based instead of acetic acid/methanol-based. Have you considered western blotting, just to check if your protein is present in only miniscule amounts? As pointed out above, silver staining is an option, but I would add SYPRO orange staining also as a sensitive alternative.

Also: check your wells for staining - it's possible your protein doesn't enter the gel in the first place, although very rare.

Hope this helps,

Arne