Keating:Cysteine conjugation

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Protocol for Cysteine Conjugation
this protocol is specific for the molecules to which I conjugate cysteines, but could be applied to any molecule
 * written by Nora

protein for conjugation with iodoacetamide (IA) for conjugation with fluorophores
 * resuspend lyophilized protein from NiNTA elution in water, measure concentration via Edelhoch, aliquot to 100 nmol in tubes, and lyophilize
 * resuspend a 100 nmol aliquot in 1 ml 1xTBS (50 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA) + 5 M GuHCl for 100 μM
 * reduce protein by adding 2 μl of 500 mM TCEP to each sample for 1 mM
 * heat 5 min, incubate RT 1 hr
 * IA - VWR 80055-298, 185 g/mol
 * want 20-200 μmol IA for 100 nmol of each protein
 * resuspend enough IA for number of samples in 1x TBS + 5M GuHCl to 1M
 * add 2.4-240 μl IA to each protein for final conc. of 20-200 mM
 * incubate 2 hr RT rocking
 * purify by HPLC
 * fluorescein maleimide (fluor/F) - Invitrogen F150, 427.37 g/mol
 * want 1μmol fluor for 100 nmol of each protein
 * resuspend enough fluor for number of samples in DMF for 20 mM
 * add 50 μl of fluor to each protein for final conc. of 1 mM
 * Alexa Fluor 568 maleimide (Alexa/A) - Invitrogen A20341, 880.92 g/mol
 * want 0.5 μmol Alexa for 50 nmol of each protein
 * resuspend 1 tube (1mg) in 113.5 ul DMSO for 10 mM (good for two proteins)
 * add 50 μl of Alexa to each protein for final conc. of 1 mM
 * incubate 4 hr RT rocking
 * use Pierce Zeba 5 ml desalting column (cat # 89892) to remove unreacted fluorophore
 * rinse columns 2x with 2.5 ml 1x TBS + 5 M GuHCl
 * add each sample to a separate column
 * rinse tubes with 100 μl (Alexa) or 200 μl (fluor) 1x TBS + 5 M GuHCl and add to columns
 * determine concentration of samples at 280 and 490 nm (fluor) or 575 nm (Alexa) by measuring spectrum from 650 or 520-240
 * take out 5 μl to run on a gel and 10 μl to run on analytical HPLC
 * aliquot in 5 tubes of 10-20 nmol each according to fluorophore concentration and rest in another tube and freeze at -80°C