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

protein

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

for conjugation with iodoacetamide (IA)

for conjugation with fluorophores

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