Anyone use Alexa Fluoro or similar reagents for HPLC/FLD?
Posted: Wed Oct 17, 2012 8:08 pm
I have been interested in tagging glycoproteins to increase their sensitivity by fluorescence detection. I assumed that Alexa Fluoro or similar reagents could be used to tag proteins such as insulin or IgG or glycoproteins such as hemagglutinin but have had no luck. Following their protocols or previous publications for labelling these type of proteins for flow cytometry, microplates, or capillary electrophoresis, it basically comes down to chromatography to resolve them. So far I have been using a UPLC C4 column with water (0.1% TFA) and acetonitrile (0.1% TFA) and running a gradient from 95% to 5% but I don't detect anything. I even look at the samples on a fluorescence microplate to make sure the published excitation and emission wavelengths were correct. It is hard to find a lot of literature of HPLC (UPLC) with fluorescence detection since it isn't widely used in industry and a search on google is flooded with glycan analysis.
I know a lot of chromatography experts but no fluorescence detection experts. I am interested in looking at the intact protein or at the least reducing it but I can't seem to get the tag to derivatize according to literature/protocols using different detection methods but similar scope. When I run the PDA in parallel, I can see my insulin or IgG eluting which would appear that the tagging didn't work. Has anyone actually used these fluorescent dyes for use in LC/FLD and have suggestions? Thanks!
It is possible that the tagged product is precipitating in the increasing acetonitrile? One thing to note, I don't even see the excess fluorescence dye eluting in the separation. With other FL derivatization reagents, I have always seen a large peak where the excess elutes but I don't see it using the Alexa Fluoro dye.
I know a lot of chromatography experts but no fluorescence detection experts. I am interested in looking at the intact protein or at the least reducing it but I can't seem to get the tag to derivatize according to literature/protocols using different detection methods but similar scope. When I run the PDA in parallel, I can see my insulin or IgG eluting which would appear that the tagging didn't work. Has anyone actually used these fluorescent dyes for use in LC/FLD and have suggestions? Thanks!
It is possible that the tagged product is precipitating in the increasing acetonitrile? One thing to note, I don't even see the excess fluorescence dye eluting in the separation. With other FL derivatization reagents, I have always seen a large peak where the excess elutes but I don't see it using the Alexa Fluoro dye.