Varsha, it could be a simple sensitivity issue - the protein concentration might be below the detection limiit of Coomassie, but still well enough to cause the activity. Trying silver is a good idea then, better would be a good western, another alternative (if available) cutting and in-gel-digesting the MW range of interest (assuming you are using MW markers and know which range to look) for high-sensitivity LC/MS/MS analysis.

It could perhaps be a concentration issue. Maybe your protein of interest is very diluted in the supernatant that you analysed on SDS-PAGE and therefore does not yield a clearly visible band. You could perform a TCA precipitation on the supernatant and then redissolve the proteins in a much smaller volume before applying the sample to SDS-PAGE.

You may have observed good activity, but that does not mean that the protein concentration is high enough. Usually activity improves after the concentration of protein starts dropping during various steps of purification. You need to concentrate the protein by either using solid sucrose during dialysis or by ultracentrifuging it. Ideally ion exchange chromatography should help. Good luck.

Thank You so much for the suggestions. I did not know about the TCA precipitation.Thank you.

Concentration is the most important reason. What is the size of your protein? You can also vary the acrylamide or bisacrylamide concentration, if your protein is very small..

SDS-PAGE is not very sensitive. As others have said it's probably a concentration issue. Staining with Silver nitrate is the most sensitive way to detect proteins but is a bit expensive and takes lab time. Failing that you could use a SYPRO which is quicker (but expensive) or if you’re using coomassie you could try adapting your method to a "blue silver" method as below:

http://www.ncbi.nlm.nih.gov/pubmed/15174055

Good luck!

final concentration of 2 mg/ml protein with 1% SDS, 10% glycerol, 10 mM Tris-Cl, pH 6.8, 1 mM ethylene diamine tetraacetic acid (EDTA) is used.

Better you go for quantitative estimation of extracted protein by Bradford/Lowrys Method and eventually follow the protocol.

concentration is necessary for SDS-PAGE.

Hi, Use most appropriate method for protein precipitation like Acetone, TCA, Ammonimum sulphate.....then check its concentration...... it is also important the sample preparation for SDS-PAGE......

A good alternative for silver staining is zinc negative staining... Its very fast and quite sensitive.. http://www.sciencedirect.com/science/article/pii/S0003269704001460

You never mentioned if you positive control for western was good or not. If it was not, maybe something went wrong in your western. If your positive control was good, then your protein could be of low concentration and you can concentrate it as highlighted above. All the best.

You need a much more elaborate description of your problem because there are way too many variables to account for with the information you have provided. For eg. are you using antibodies to detect or simple comassie staining in that case it could be a problem of your detection reagents, what are your controls and this is very important without this you are all at sea, however i completely disagree that western blot is not sensitive infact it is very sensitive down to ng quantities of the protein unless ofcourse you have a protein with super high specific activity i.e. even 1-2 ng of protein is enough to register as activity on your actvity assays which brings another question to the fore i.e. what is the sensistivity and specificity of the activity assay that you are using and could it be possible that you are getting the activity as a background(which will be solved ofcourse if you have a negative control and hence the requirement for proper controls). I think you should provided a more detailed question.

I have herewith sending the protocol and Troubleshooting guidelines for SDS and Native PAGE. Find the attachments..

Do a Bradford assay, get the protein concentration (ug/ul) in your sample and then load different amounts (10ug, 25ug, 50ug) of your proteins in your gel and run it. That way you can see if you are using too little concentration or not.

Could it be a possibility that your protein of interest isn't being expressed in the cell?

I hope this helps.

Correct. Let us know if your positive control for western was good or not. If it was not, maybe something went wrong in your western. If your positive control was good, then your protein could be of low concentration and you can concentrate it as highlighted above. All the best.

Some proteins do not show up with Coomassie, whatever the concentration. Another argument for trying silver or (better) western-blotting if you have an antibody.

yes i had used control as well along with this strain.I found nice bands in control but not in my strain.I am using coomassie staining but now i am also trying silver staining..

Varsha, it could be a simple sensitivity issue - the protein concentration might be below the detection limiit of Coomassie, but still well enough to cause the activity. Trying silver is a good idea then, better would be a good western, another alternative (if available) cutting and in-gel-digesting the MW range of interest (assuming you are using MW markers and know which range to look) for high-sensitivity LC/MS/MS analysis.

I had performed silver staining, when i incubated the gel in developing solution the color didn't developed in the middle of the gel.what might be the reason?

Let us first agree that when we get a very high activity of a particular protein, then in all likelyhood the protein concentration will be very low and may not appear in SDS PAGE. Here the solution is to concentrate the protein by dialysing it against solid sucrose and then run the PAGE.

There is a fundamental difference between an 1) activity assay in a complex sample (secreted protein in culture medium) and a 2) protein gel. Biological activity can be much more sensitive compared to total protein stain (Coomassie, or Silverstain, or Ponceau red after blotting) or even a western blot if the antibodies are not very good. More important, the biological activity can originate from several proteins that are isoforms, with slightly different molecular weights, making it difficult to discern a specific band. The control experiment to find out where you stand is to have a positive control in the form of your purified target protein, you can then do dilution series and check the detection limit for the biological activity assay, western blots or unspecific stains that bind to all proteins. In general, I am not surprised that you should have this issue, most enzyme essays are much more sensitive than protein stains or antibodies (the antibodies would have to be of superb affinity and titre to beat an enzyme essay), but it depends on case to case.

I am having a similar experience with my pectinase. Faint band was noticed at high concentrations of my sample though an improvement but not intense enough for my liking. I want to consider trying silver staining.

probably the protein you loaded was not enough. try silver staining if you want a more sensitive technique

I have the same problem, unable to see the protein bands in my gel. I thought it could be due to the concentration issue but even I increased the concentration I faced the same problem. I even lyophilize the samples.What could be the possible reason?