protein - protein interactions

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g a
g a's picture
protein - protein interactions

Hi all
I am working on the kinetics of the carrier protein binding to small molecular proteins and peptides..... Does any1 have experience in handling the uv spectroscopy or CD spectra..... Will it be useful for this kind of work..... I mean the spectra will show varaitions in binding if a pure protein is treated with binding peptides but in my case i have a fraction of proteins which is bound to the carrier protein but then......how can i study kinetics of protein interactions in this case  ........... I have very little idea and Im relatively a beginner in the field....
we also dont want ot use detergents or urea or acetonitrile for the protein protein disruption work....... especially detergents......
Kindly help if you can
Thanking you in anticipation.
Gaganjot Singh

RLS
RLS's picture
Have you considered

Have you considered fluorescence anisotropy? I will pull up some papers that have great info on the technique (check out publications from Michael Ibba's lab. He's at Ohio State University).
Hope this helps!
Lynn

martin wang
martin wang's picture
Surface Plasmon Resonaces

Surface Plasmon Resonaces technology might be an excellent choice for your application. You are then able to monitor and record the binding kinetics without manipulate the samples, label free real-time.
You can anchor one protein on the sensing chip, and just have the analyte flow through the system. Biosensing Instrument makes a low cost, quite flexible system.

g a
g a's picture
thank you for the suggestions

thank you for the suggestions....
Lynn I will be looking forward for the paper references.....
Thanks again.
Gaganjot Singh

Hatuey
Hatuey's picture
Hi, yes you can effectively

Hi, yes you can effectively use anisotropy as was suggested; for this you need to label the small peptide with a fluorescence dye , upon binding the mass of the labeled peptide will increase and the motion or rotation of the molecule be slower, and this changes the polarization of anisotropy, however, if the change in mass is not large enough, the technique might not be sensitive enough to be detected. So, I suggest you consider FRET. if by chance you have tryptophans in either the peptide or the protein, for example, you have tryptophans, one or more, in the peptide, but not in the protein, or viceversa, then you can label the protein or the peptide with a dye that quenches or is an aceptor of the tryptophan fluorescence. This is a more straightforward method of quantifying binding and the kinetics of binding . You can find more information about FRET in Molecular Probes or Anaspec web sites. I f you dont have Ws in either or there are Ws in both then you need to label both the peptide and the protein. Hope this helps.

g a
g a's picture
We dont have facility or

We dont have facility or expertise for carrying out FRET. However i can carry out UV or IR spectroscopy. How much help would that be in such kind of work. Mine is not even 100% pure protein its almost 90-95% purity.
 

Hatuey
Hatuey's picture
Hi again, Another option I

Hi again, Another option I would consider then is to buy crosslinkers from Pierce or a different company to find if there is binding between your proteins, all you need is to incubate the proteins with the crosslinker and run a gel afterwards. This doesn't give you kinetics though, just if there is binding or not.

RLS
RLS's picture
 Hi,

 Hi,
While you may not have the equipment, here is the info:
These labs' websites can be quite useful for general information:

http://www.yale.edu/miranker/Research.html
 
http://www.yale.edu/reganlab/research.html
 Here is a link to an invitrogen website that is useful:

http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Polarization-FP.html

Lastly, here is a paper that uses the technique:

Mette Prætorius-Ibba, Corinne D. Hausmann, Molly Paras, Theresa E. Rogers, and Michael Ibba. (2007) Functional Association between Three Archaeal Aminoacyl-tRNA Synthetases. J. Biol. Chem. 282:3680-3687.
 Best of luck in getting your experiment to work!
Lynn

 

 

 

qiang wang
qiang wang's picture
ITC will  give the binding

ITC will  give the binding kinetics of your interacting proteins.
if you do not have one, run CD spectra every couple of minutes allows you see the binding kinetics by changing spectras, but you will then need to find a way to explain you spectra