I expressed an enzyme and checked their activity at two fraction, extracellular and intracelluar. Both have certain enzyme activity but when I run on SDS-PAGE, both did not show any expected band. How to solve this?
Are you using an antibody for the detection of your protein or a protein stain (i.e. coomassie)?
In that case, it might be usefull to fix your gel before staining as this can help prevent the protein from diffusing out of your gel prior to staining.
Maybe have a look at:
https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314716762536/litdoc80634334AB_20110830181131.pdf
It sounds to me that the expression level of your protein of interest is just low.
In a whole cell lysate, you have to have a certain level of expression of your protein before you can see it above the background bands. For extracellular, your protein is going to get seriously diluted into the culture media, and (unless expression is very high) can easily be too dilute to see by coomassie staining.
Do what the others have suggested, and run a Western blot with a protein or tag specific antibody. Alternatively, concentrate the protein from the media by e.g. TCA precipitation and re-run your gel. Another possibility is, since you know you have activity by assay, simply pass your two fractions over an appropriate column (e.g. a nickel column, assuming you have a His-tag) and then look for your band in what will now be enriched fractions.
Are you sure Coomassie stains your protein? Coomassie can only stains something like 95% of proteins (possibly due to protein composition), so if you're not positive Coomassie can stain your protein (especially in its current/expected form), I'd suggest trying another stain
Try silver staining the gel. Its relatively easy to do and has a much lower detection limit than Coomassie. If you need to use your gel for other downstream applications colloidal stain is another alternative, however it still does not have the detection limit of silver... As many people have mentioned, I think your main problem is concentration...
It looks as a problem of concentration. All stains have a critical protein quanity that they can recognize. If you want to stick to Coomasie stain you could try using colloidal Coomasie stain instead, as it is more sensitive. Since your protein is an enzyme, it only takes a very small quantity of it to see enzyme activity but it needs a bigger concentration for the gel.
However, as David Lloyd mentioned you could use a silver stain procedure. Silver stain is far more sensitive than colloidal coomasie. Although, keep in mind that when working with silver stain one has to be careful with tha handling of the gels, so as not to get black smears on the gel itself.
Another possible solution, could be fluorescent dyes that are used for staining 2D gels. They have a very good sensitivity, however they are more pricey.
Good luck with your experiment!
Most likely is a problem of concentraton. you should try to add more protein (known amount) and I believe that the untransfected or mock transfected cells were negative for the enzymatic activity. If there is an antibody, as other people mentioned, go for immunodetection. Otherwise you can either use a tag (HA is easy to make and the antibody works wonderfully) or even a GFP fusion.
If you are expressing your protein in E. coli, you may have a tag based on the plasmid. Many of our expressed proteins have a his-tag, that can is used to purify the protein. Now you can use an anti-his antibody to detect your protein through Western. The availability of the his-tag can also be exploited to purify your protein to near homogeneity, which will overcome the concentration issue raised by others in this blog. Some plasmids can have the flag-tag, that can be used in a similar manner.
Hope this helps
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