A clinical chemist wants to use a fluorescently labeled antibody for staining a

Question

A clinical chemist wants to use a fluorescently labeled antibody for staining a cancer biomarker in

histological tissue sections. Anti-Her2 (an antibody targeted to Herceptin-2, a protein with diagnostic

value in various cancers) was reacted with the N-hydroxysuccinimidyl (NHS) ester of

carboxyrhodamine dye (?530 nm = 116 000 M

A clinical chemist wants to use a fluorescently labeled antibody for staining a cancer biomarker in histological tissue sections. Anti-Her2 (an antibody targeted to Herceptin-2, a protein with diagnostic value in various cancers) was reacted with the N-hydroxysuccinimidyl (NHS) ester of carboxyrhodamine dye (?530 nm = 116 000 M-1 cm-1). NHS esters react with the amine side chains of lysine residues in proteins. After the reaction, the anti-Her2 (?280 nm = 210 000 M-1 cm-1) was purified from excess dye using size exclusion chromatography. The absorption spectrum of the final conjugate was measured in a cuvette with a 1.00 cm path length. Determine the average number of dye molecules per antibody from the spectrum in the spreadsheet accompanying this problem set. A reference absorbance spectrum for a 1 ?M solution of the dye alone is included. All spectra are background corrected. The antibody solution does not have any visible absorbance.

Explanation / Answer

ans : 8 dye molecules per antibody

reference dye molecule abs spectra shows 0.1000 anti-her gives 0.8000 which implies 8 dye molecules

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