[evidenso]

Hello
I'm doing a master thesis: Raman spectroscopy of C-reactive protein.
I have no chemical baground at all. So I would like to hear if any of you chemists can recommend a book of protein handling or show where on the internet i can find info. It have to answer quistions like: Storage, what happends when the bottle with protein has been opened. How to handle it. and so on   

[Yggdrasil]

Current protocols in molecular biology or others in the current protocols series is a good resource.

http://www.currentprotocols.com/WileyCDA/CPTitle/isbn-047150338X.html

[enahs]

Never vortex mix your protein. Had a guy in our lab do that one time and killed ~$5000 worth of protein .

[HP]

I agree with enahs - it's not very clever to do vortex mix protein solution. Proteins first are high molecular compounds and intensive mixing (by vortex, ultrasonication ans so on) can with sure cause some bonds cleavage which can dammmage the biological function(s). And second this action can destabilize or demolish the second, third anf fourth (if the particular protein has) supramolecular structures...

[Yggdrasil]

Some more tips from a protein biochemist:

Proteins are also very sensitive to pH, temperature and ionic strength.  It is best to keep protein at 4^oC until right before you need it.  Never freeze protein.  If you wish to store it for extended periods of time, you can try making a solution in 50% glycerol and storing at -20^oC, or flash freezing the protein in the standard buffer and storing at -80^oC (some proteins won't survive flash freezing however).

Always dissolve your protein in buffered salt solution (conditions depend on your exact protein, but pH 7-8 and 100-150mM NaCl are pretty standard conditions).  Dissolving your protein in a solution with the wrong pH or with the wrong ionic strength (e.g. pure water) can inactivate your protein.

[evidenso]

OK thank u very much, great help. You say that pH is a great factor, Im sure it is, as it changes the electronic UV spectrum of the molecule. Do you know why this is? Can you recommend a book which descreibe why pH and for example polarity changes the proteins states?

[Yggdrasil]

From this site (http://www.ruf.rice.edu/~bioslabs/methods/protein/abs280.html)

Principle
Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm. Secondary, tertiary, and quaternary structure all affect absorbance, therefore factors such as pH, ionic strength, etc. can alter the absorbance spectrum.

It is definitely true that the 200nm absorbance is primarily due to the amide bonds and the 280nm absorbance is primarily due to the aromatic side chains (phenylalanine, tyrosine, tryptophan), but I wouldn't really expect changes in the conformation of the protein to change the absorbance spectrum much.  I know restricting the rotational freedom of some fluorophores can affect the intensity of their emission, but I don't know of any effect that exists for absorption.  Perhaps you might have a better idea.