DC271

Guide (The following is our recommended solution. This solution is merely a guideline and should be modified according to your specific needs.)
DC271 Fluorescence Competition Binding Assay for CRABP-II Binding Experiment
1. Reagents and Solution Preparation
1.1 DC271 Stock Solution: Dissolve DC271 in anhydrous DMSO to prepare a 10 mM stock solution.
1.2 CRABP-II Protein: Recombinant expressed and purified human CRABP-II (or CRABP-I), dissolved in an appropriate buffer (such as 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM DTT), concentration determination (A280 or Bradford).
1.3 Detection buffer: Low fluorescence background buffer, recommended 20-50 mM Tris-HCl, pH 7.2-7.5, containing 50-150 mM NaCl.
1.4 Test compound (competitive ligand): Dissolved in DMSO, with the final DMSO concentration in the system ≤ 1% (typically ≤ 0.1%-0.5%).
1.5 Blank control: Equal volume of DMSO; Positive control can be ATRA (HY-14649) or EC23 (HY-12309).
2. 96-Well Plate Fluorescence Replacement (Competitive) Experiment
Suitable for qualitative initial screening or quantitative determination of IC₅₀/Kd:
2.1 Add CRABP-II + DC271 complex In a 96-well plate with black color, low adsorption and non-binding surface, add:
CRABP-II: final concentration 100 nM
DC271: final concentration slightly higher or equal (also commonly 100 nM - 200 nM, to ensure saturation binding)
Make up the volume with the detection buffer (usually final volume 100 - 200 μL/well), gently mix.
2.2 Formation of DC271-CRABP-II complex
Incubate at room temperature in the dark or at 4℃ for 10–30 minutes to allow the binding of DC271 and CRABP-II to reach equilibrium (the fluorescence signal reaches a plateau).
2.3 Adding Competitive Ligands (Test Compound)
Add the gradient-diluted test compound or a single concentration (for qualitative screening), mix well, and incubate in the dark for 15-30 minutes.
2.4 Fluorescence Detection
Using an enzyme detector for testing: Excitation (Excitation): 355 nm (within the range of 340 - 410 nm) Emission (Emission): 460 nm Subtract the blank value of the buffer solution, and record the fluorescence intensity (FI) of each well.
2.5 Data Analysis.

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