[kriggy]

Hi I have some questions and I cant find any suitable aswers:

a) I found that total proteins from purple complex with Cu ions in alcaline condition and then the amount of protein is determinated by spectrophotometric analysis of that product. Does the method account for one protein coordinating  multiple Cu ions? Or does it matter that multiple Cu ions are coordinated? (I think the more Cu ions the greater absorbtion of light) Are there any other methods ?

b) One can determine Mr of protein by MALDI-TOF MS. I understand that MALDI is soft ionization method which gives little fragmentation so its possible to see the (pseudo)molecular ion?
The ion trap detector we have at school at our HPLC-MS has range of m/z 100-2000. I was told that its possible to analyze proteins on this device too because the peptide chain can bear multiple charges but if for example I see peak with m/z=1000, how can I know whether its 1000/1 or 2000/2? Does it mean that every atom that could be protonated is protonated? That seems very unlikely..
what are other methods for this? Gel electrophoresis with suitable standards of known Mr?

Thanks for help / hints

[Babcock_Hall]

With respect to your question in (a), more than one method uses copper ions.  For example, a common assay that is more recent that some other methods uses bicinchoninic acid (BCA).  Another method is the Biuret assay.  Which method are you using?
http://www.ruf.rice.edu/~bioslabs/methods/protein/bca.html
http://www.ruf.rice.edu/~bioslabs/methods/protein/biuret.html

[kriggy]

None of them, im revising for biochem exam and part of one of the questions is "methods of total protein determination" and "determination of Mr of protein"
Thanks for links, I will check them

[Babcock_Hall]

With respect to (b), can you think of any protein chromatography techniques that separate on the basis of molecular mass (approximately)?

[Arkcon]

The coordination of Cu ions to various protein molecules does differ from protein to protein.  To quantitate, you will need to generate a standard curve for the protein in question, or at last identify what protein you used to generate a standard curve -- i.e. you use BSA, and say "protein conc relative to BSA standard curve"

[kriggy]

With respect to (b), can you think of any protein chromatography techniques that separate on the basis of molecular mass (approximately)?

gel electrophoresis?

[Babcock_Hall]

Gel electrophoresis is not exactly chromatography, but it is similar.  Most gel electrophoresis of proteins is done under denaturing conditions.  How about a chromatography technique that is often used on native proteins during the purification process?

[kriggy]

Ok the only two I can think of are GPC or affinity chromatograpy. But Im not sure if its this one. It can separate the proteins by their mass but how do you determine the actual mass without using MS?
And affinity chromatography can separate the protein I want but wont tell me anything about its size.

[Babcock_Hall]

You might want to look into size-exclusion chromatography, which is sometimes also called gel filtration chromatography.