Maleimide-DOTA (Synonyms: Maleimido-mono-amide-DOTA)

This protocol only provides a guideline, and should be modified according to your specific needs.
1. Solution preparation
(1) Working solution: Weigh the solid and dilute it to the required working concentration with the experimental buffer.
Note: The solid should be stored at -20°C or -80°C in the dark. Please adjust the optimal working concentration according to the actual situation or refer to the literature to set the gradient concentration by yourself. The working solution must be prepared and used immediately.
 
2. Experimental steps for protein labeling using Maleimide-DOTA^[1] (from the literature, for reference only)
(1)Disulfide bond reduction protein: Dissolve the protein in PBS (pH 7.4). Add 280-fold molar excess of DTT to a final DTT concentration of 20 mM. Incubate the mixture at 40°C for 30 min.
(2)Purification of reduced protein: Use a NAP-5 column to purify the reaction mixture to remove DTT. The purification column should be pre-equilibrated with degassed 0.2 M NH4OAc (pH 6.5). Collect the fractions containing protein.
(3)Binding with Maleimide-DOTA: Add 11-fold molar excess of Maleimide-DOTA (dissolved in 0.2 M NH4OAc, pH 6.5) to the purified protein. Incubate the mixture at 40°C for 1 h.
(4)Purification of bound protein: Purify the reaction mixture using a NAP-5 column to remove unbound Maleimide-DOTA.
(5)Protein concentration measurement: Measure protein concentration using a DS-11 spectrophotometer.
(6)Storage: Store the bound protein in 0.2 M NH4OAc (pH 6.5) at −20°C.
 
Note: This experiment provides a reference protocol for protein labeling using Maleimide-DOTA. The reaction concentration and reaction time can be optimized according to actual conditions. The operation should be performed in a sterile environment to prevent contaminants from interfering with the reaction. Avoid direct contact with the reaction reagents.
 
References:
[1] Deyev S M, Xu T, Liu Y, et al. Influence of the position and composition of radiometals and radioiodine labels on imaging of Epcam expression in prostate cancer model using the DARPin Ec1[J]. Cancers, 2021, 13(14): 3589.