I am trying to immobilize peptide/proteins onto the surface of PCL (polycaprolactone) by making use of EDC/NHS chemistry. Firstly, I want to confirm whether the conjugation was successful. Secondly, I want to determine the amount of peptide/protein that has been conjugated onto the PCL surface (for eg. ug/cm2). What are the methods which will be helpful for the same? I would really appreciate your help.
You can do it by using UV-vis, FTIR, XPS and SEM analysis. Please see the attached papers:
Article Immobilization of proteins onto novel, reactive polypyrrole-...
Unfortunately, this question is not this easy as it seems. Nearly all methods are semiquantiative or even qualitative at best. If you are working with particles, it might be the most accurate method to hydrolyze the protein on the surface by acid or base and to determine some or all amino acids with a suitable calibration:
Article Protein Quantification by Derivatization-Free High-Performan...
Article Quantification of hydrolyzed peptides and proteins by amino ...
Surface sensitive techniques will answer your question. For instance QCM-D or SPR would help where in you would study the adsorption/chemisorption reaction in-situ. PCL also has Nitrogen in the form of Amide, so XPS analysis may not be straight forward. Sttill there is a possibility, or instance of cysteine groups could contribute to Sulfur peak. So as the coverage increases the sulfur peak might increase. Also the protein might hold on to counter cations Na/K which could give peaks. In this case the washing has to be performed quite carefully, especially the pH of MQ/DI water should always be the same. If that fluctuates a lot, the counterions may be lost.
Also an indirect way would be water contact angle. Depending on the PI of your protein and also the pH of water used, the CA would change with coverage of protein and would stabilize after monolayer (enough to exclude the PCL surface underneath) coverage . This wont be quantitative.
Thanks everyone for their valuable inputs. I have pretty much figured out the qualitative methods. Currently, I am trying to conjugate BSA protein to PCL scaffolds to optimize the parameters for EDC/NHS chemistry. When I dip my scaffold (post EDC/NHS conjugation) into the BCA assay, it turns blue so I am thinking of using the indirect method for quantification of protein conjugated using some spectrophotometry.
Also, if I tag the BSA protein with some fluorescent dye like RBITC, FITC, etc and try to correlate the fluorescent intensity with the protein surface coverage i.e. conjugation, would that work?
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