Wow! A very open ended question, the answer to which depends massively on what you want to know about your protein, and what your protein actually is!

With a TM protein, one might not be able to simply throw it at cells and expect it to integrate with the membrane properly, so sadly this option is perhaps less useful.

However - In vitro functional tests, such as ligand binding assays perhaps?

Biophysical characterisation maybe? SEC-MALLS, AUC, CD spectroscopy, may help you better understand the protein.

You may get a more focussed answer if you provide a little more information about your TM protein - it is a receptor? A transporter?

Wow! A very open ended question, the answer to which depends massively on what you want to know about your protein, and what your protein actually is!

With a TM protein, one might not be able to simply throw it at cells and expect it to integrate with the membrane properly, so sadly this option is perhaps less useful.

However - In vitro functional tests, such as ligand binding assays perhaps?

Biophysical characterisation maybe? SEC-MALLS, AUC, CD spectroscopy, may help you better understand the protein.

You may get a more focussed answer if you provide a little more information about your TM protein - it is a receptor? A transporter?

You could check for purity by biophyical methods or by functional tests.

For crystallization you should try the addition of ligands.

Have you tried crystallization attempts in cubic phase?

As David has mentioned in his post, it's an open ended question. Problems in science can only be solved under some contrains. If you could let us know more about protein the answers can be more realistic. if not characteised you can study ligand-binding kinetics by ITC or SPR!!!

I think you should go for sequencing followed by bioinformatic tools that will unfold many of its characters like conserved regions, homology with other known membrane proteins etc etc.....

Go it for crystallization by freezing and do data collection for thorough investigation for its structure and then search and match it for protein crystallographic data from a data bank (One at IISc Bangalore) for its characterization and study its properties.

Your statement assumes that the protein is PURE and is in DETERGENT MICELLES. I also assume that the primary structure is known so you are absolutely familiar with the protein itself. You can do a lot of biophysical (mostly spectroscopy) studies already on the detergent-protein micelles. However, you can try reconstitution in proteo-liposomes and study some trans-membrane properties of the TM protein. To perform such experiments you need "substrate-loaded" proteo-liposomes.

let us know a little more about your TM protein (and the detergent that keeps your TM protein in solution).

One can do lots once you have purified the protein.. since its a TM protein.. so u can determine its 3d structure... can do an evolutionary study using phylogenetics.. or some kinda structural work using XRD or NMR...

If it is an unknown protein, you could sequence it. If it is known, you can study its thermodynamics (e.g. DSC), aggregation kinetics (e.g. light scattering, UV-Vis spectroscopy, rheology), conformational changes (e.g. SAXS, CD), binding with partners (e.g. SPR, AFS), electronic properties (e.g. STM, CS-AFM) and much more...

Depends on the experimental facilities available and on the function fo the protein. A number of neat experiments can be planned.

I agree with Sheraz, sequencing the protein to confirm the identity of the protein you expressed. This is simple and rapid way to confirm. Then move on to some of the other experiments. With purified protein, you can evaluate ligand binding etc.

With a pure protein, you can expect to do lots of things.

(1) You can try to further purify it to absorbance 260/280 ratio below 0.8, and try to crystallize your protein. The more free your protein is from contaminants, the more probable that you end up with a clean crystal. This way, you try to determine the structure of protein and be in forefront of being the first person to solve the structure by publishing a paper. Sounds too tempting.

(2) You can design molecular biology experiments so as to understand its interactions with DNA/RNA (based on what the protein does) or any important molecule. You can analyze the various features of your proteins by studying its interactions with biological molecules.

(3) You can use bioinformatic approaches to find the closest homologue in any species and try to relate your protein of interest to that described in literature. Sometimes, the evidence about protein interactions by molecular biology data may not correspond to structural biology data. You can dig more in this aspect and may end up finding a never-before known molecule that plays interfering role. (who knows, anything is possible in biology).

(4) You can even think of clinical trials in this perspective, wherein, your protein may be useful for a set of patients with a syndrome or disease.

The question asked is wide open and you can do smart things with pure protein. One can also think of commercializing these proteins at industrial scale for economic benefits.

Hope this helps,

Regards,

Sagar Sridhara

More information about the protein would help us to advise you, but other thoughts which come to mind that haven't yet been mentioned:

Does it have any activity - enzymic, binding? If you do the bioinformatic searches which have been recommended you may find related proteins with activities you can try assaying your protein for.

Does it have channel activity - if it assembles into a helical bundle or a porin-like beta barrel (and depending on your bioinformatic searches), you may want to see if it acts as an ion (or similar) channel. If you suspect this, I'd talk to electrophysiology people who do single channel recording/patch clamping for advice.

Dominic

First of all i would like to say thankyou to all of you for your valuable suggestions. Special thanks to Sagar Sridhara,Alajos Bérczi and David Brigg sir. Most of them has suggested me for Biophysical study, Spectroscopy, ligand binding analysis and other bioinformatics approches for better understanding our protein. All these suggestions are appreciable and now i am crosschecking my protein that what are approaches i can use. Some of them have asked some more details about my protein, Here i would like to add description for those scientists that My protein (Neutral Sphinglomylinase) belongs to hydrolase family which is responsible for breaking down the sphingolipid into phosphocholine and ceramide.

It has five types 1) Acid Sphingomylinase (SMase) 2) Neurtal SMase 3) Alkaline SMase. Here Acid and Neutral has one-one subtype. I am working on Neutral SMase for which currently no crystal structure avaulable for human and other closest species. Currently there is 4 Crystal strucutre available at PDB for neutral sphingomylinase of Baccilus cereus. First i thought to check the protein sequence by sequencing method but i am not getting lab to these.If any one can suggest me for some suitable lab in India where i can get sequence of my purified protein, it will be appreciable. Other approches will open after geting the clear crystal and diffraction data for which i am working..Once again thank you all for your valuable suggestions.

you can do sequencing at IIT mumbai, can help you in this regard by providing contact details of the concerned lab and personel ...

Thank you Sheraz for your information...I will try to contact their lab.

Native gel to see how many bands it gives there

Iso-electric focussing to determine real pI

Pleasure om... Good luck with it