AF568 C5 Maleimide

Guidelines (The following is a recommended experimental protocol for guidance only and should be adjusted according to your specific needs).
1. Preparation and Reagents
1.1 Protein/peptide: Dissolve in degassed buffer free of exogenous thiols at a concentration of 50-100 μM (1-10 mg/mL).
1.2 Reaction buffer: 10-100 mM phosphate, HEPES or Tris, pH 7.0-7.5.
1.3 Dye stock solution: Reconstitute AF568 C5 Maleimide using high-quality anhydrous DMSO or DMF.
1.4 Reducing agent (if needed): Use TCEP (HY-107859) (100-fold molar excess for disulfide bond reduction), and ensure complete removal of or avoid using DTT (HY-15917)/2-Mercaptoethanol (HY-Y0326), as they quench the reactivity of maleimide.
2. Conjugation Procedure
2.1 Thiol reduction: For buried disulfide bonds, treat the protein with 10-100-fold molar excess of TCEP at room temperature for 20-30 minutes under an inert gas atmosphere.
2.2 Dye addition: Add 10-20-fold molar excess of AF568 C5 Maleimide stock solution to the protein solution.
2.3 Incubation: Mix thoroughly and react at room temperature for 2 hours, or react overnight at 4 °C in the dark under gentle shaking.
2.4 Quenching (optional): To stop the reaction at a precise time point, add a small molar excess of exogenous thiol-containing amino acids (e.g., L-Cysteine (HY-Y0337) or DTT) to terminate the reaction.
3. Purification
Separate the labeled conjugate from free unreacted dye using a gel filtration column or a centrifugal ultrafiltration tube with a molecular weight cutoff of 10 kDa.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.