Modifying protein amino groups with fluors
Fluorescent amino group reagents
Dansyl chloride is the most generally useful; pyrenesulfonyl chloride is also useful. They react with protein amino groups to give fluorescent derivatives. Detection with midrange UV light sources is about ten-fold more sensitive than Coomassie Blue staining, but about ten-fold less sensitive than silver. Fluorescamine, o-phthalaldehyde, and 2-methoxy-2,4-diphenyl-3(2H)furanone (MDPF) fluoresce only after reaction with amino groups. Of these only MDPF is stable and resistant to photobleaching. Proteins to be run on SDS gels should be reduced and alkylated prior to amino group modification.Dansyl chloride modification of proteins for SDS PAGE
Spectral properties of dansyl derivatives
1- Dissolve protein in borate cracking buffer.DTT (M=154.2) - 77mg + 0.95ml DW
2- Add 0.02 vol of 0.5M dithiothreitol (fresh), boil 1.5 min. (see DTT)
3- Add 0.09 vol of 0.25M iodoacetamide and heat 1.5 min at 100C.
4- Add 0.1 vol of DNSCl (5 mg/ml in acetone). Incubate 5 min at 75C.
5- Electrophorese aliquot.
6- Following electrophoresis, examine gel with midrange UV light.
IAm (M=185) - 47mg + 0.98ml DW
Iodoacetamide and dansyl chloride are light sensitive and should be stored in brown tubes.