I use EDC-NHS procedure to covalently bind an RGD peptide on PLGA nancapsules. I tried Micro-Bradford Essay but in literature coomassie dye binds to NH2 terminals of arginine histidine and lysine, which EDC-NHS uses to form amide bonds with carboxyl groups of PLGA. In this case coomassie reagent could not react and will not be useful. I have seen that BCA essay is also used but my sequence does not have tryptophan, cystein or tyrosine. Does anyone has any suggestions to quantify the amount of RGD peptide bound to PLGA capsules? (By the way I tried FTIR/ATR but it seems sensitivity of the device was insufficient for such small amounts of proteins)
HPLC is the most common approach I have seen for quantifying RGD conjugation to polymers or lipids. I have had good results with Fluorescamine quantifying similar protein conjugations to PLGA spheres. Fluorescamine only produces a response when it interacts with free amine groups (which are present on RGD) so this may be a simpler assay for quantifying how much RGD is on your PLGA surface. You just need the a fluorometer with appropriate filter sets.
Paper about fluorescamine:
http://www.biotek.com/resources/articles/fluorimetric-quantitation-protein-fluorescamine.html
Paper that measures RGD conjugation to PPO-PEO block polymers
http://pubs.rsc.org/en/content/articlepdf/2011/SM/C0SM00612B
You can try indirect quantification of peptide which means measuring the remaining amount of RGD after conjugation to polymeric nanopaticles surface. If you know the amount of RGD before conjugation and can measure the amount after conjugation, the difference between those two should be the approximate amount conjugated to nanoparticles. You need to wash nanoparticles multiple times to remove any nonspecifically adsobed RGD peptide. You can use cutoff filters to get rid of EDC-NHS from peptide solution to avoid any interference with quantification.
HPLC is the most common approach I have seen for quantifying RGD conjugation to polymers or lipids. I have had good results with Fluorescamine quantifying similar protein conjugations to PLGA spheres. Fluorescamine only produces a response when it interacts with free amine groups (which are present on RGD) so this may be a simpler assay for quantifying how much RGD is on your PLGA surface. You just need the a fluorometer with appropriate filter sets.
Paper about fluorescamine:
http://www.biotek.com/resources/articles/fluorimetric-quantitation-protein-fluorescamine.html
Paper that measures RGD conjugation to PPO-PEO block polymers
http://pubs.rsc.org/en/content/articlepdf/2011/SM/C0SM00612B
Thank you Ujwal and Shashank, we will start with indirect method probably. And Shashank I will check the protocols and if we have chemicals in hand we will try that too. I will post if we make those protocols work. I also have seen that in a paper they used proton NMR. We do not use NMR frequently, so I am not sure NMR results would be too hard to interpret.
You could still try the BCA assay, because the reduction of Cu2+ to Cu1+ is not only done by the mentioned amino acids but also by the peptide bond itself.
Be aware that if you determine your RGD by the indirect method your results wont be exact since adsorption to plastic ware etc. does also feign a higher RGD conjugation to the nanoparticle.
Fluorimetric Protein Quantification Using the Reactive Compound Fluorescamine read this article
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