Dear RG community,
I need your help!
I have a mixture of proteins attached to a surface. Some proteins are available for binding, others are not, because surrounded by other proteins. I need to identify only the proteins that are "exposed" on the surface, the ones that are available for binding. (check the attached image for clarifications)
Therefore, I need a way to label/tag only exposed proteins or amino acids, purify them, and identify them by different techniques (for example, mass spectrometry).
Any input is appreciated.
Thank you!
You can label accessible lysine residues and amino termini using a water-soluble amine-reactive compound, as long as the proteins are not in an amine-containing buffer.
Dear Valentina,
hydrophilic molecular probes functionalized with N-hydroxysuccinimidyl esters (NHS), or better still in your case: sulfo-NHS esters or STP esters (sulfo-tetrafluorophenyl) might give you some selectivity and tag only surface exposed K residues without penetrating in hydrophobic regions. (Sulfonate groups are negatively charged at physiological pH and will make the reactant more water-soluble)
perhaps choose also a probe also equipped with hydrophilic substituents on the fluorochrome core (sulfonates, chains with quaternary ammonium groups etc) such as Alexa series.
best
Cedric
Dear Adam B Shapiro and Cedric Lion, thank you for your reply! Very useful indeed. I found this simple guide that explains the process https://www.thermofisher.com/ca/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/amine-reactive-crosslinker-chemistry.html
Once labeled/tagged, how can I separate the labeled proteins from the rest? Will mass spectrometry/polyacrylamide gel allow me to analyze/visualize only the proteins of interest?
Thank you!
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