Do you have access to mass spectrometer such as MALDI-TOF? It will be the quickest way to identifying a protein.

There is one at our department, but they were not able to identify it.

2-D electrophoresis gives better resolution of proteins and you have to cut the spots from gel and digest them before mass analysis. If you know the iso electric point it will be useful for the first dimension electro focusing. If you know which family your protein or enzyme belong to then it will be easier to identify by database search. You can also perform Western blot or if you have in solution then ELISA may be the option. In this case you need suitable anibody kit.

I have no idea what enzyme is it, what family it belongs to and I have no antibodies.

Cut the band with your "purified protein" from your gel, and then find who can do the in-gel digestion + mass spec analysis. Google " in-gel digestion mass spectroscopy".

What was the aim of your project? How did you know that you have purified your enzyme without knowing anything about it?

During the purification did you use any specific assays to determine this enzyme, if so what assays? You said you want to identify within a week, have you had any luck so far?

I have measured activity in vitro, but that was all I knew. It is brand new enzyme (except the activity in vitro was described 20 years ago).

I've got some results now, so I will see what will I get after heterologous expression.

Interesting! Good luck with your project Hluska.

Thanks. However my bosses want to confirm the result because it seems too crazy to them :o)

If you are a member of Linkedin then you can post your question on the discussion groups like purification-biologics, I am sure you will receive more response. Mass spec will be the quickest way of identifying proteins/enzymes etc.

Western blotting will show the way

Excise the bands of interest from the gel, carry out a tryptic digest and mass spec finger printing analysis.