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Optimization of An FABP Ligand Binding Assay and Determination of Xenobiotic Affinities
Optimization of An FABP Ligand Binding Assay and Determination of Xenobiotic Affinities
FABP5 AEA
Yu et. al., 2014
Evidence of FABP5 Binding to Xenobiotics
THC
• THC competitively inhibit AEA binding with
FABP5
• Drug Displacement
• Drug Screening
• Method – Qualitative Decrease in Fluorescence in the Presence of Drugs
Fluorescence Assay
Method
• Purpose: determine Kd of ANS for
calculating Ki of the drug
• Well plate to set up titration
• FABP5 = 1μM (1:200)
• ANS = 1μL at varying concentrations
• EtOH = 1μL (no ANS control)
• ANS dissolved in EtOH
• add buffer to final volume of 200μL
• Set up on ice
• Between scans, plate at room
temperature
• Automix on fluorimeter
• mixing with pipet caused values to
greatly deviate
Fluorescence Assay
• No background 180
Fluroescence
1uM FABP5 + 1.8uM ANS
increasing concentration 100 1uM FABP5 + 1 uM ANS
1uM FABP5 + 1uL EtOH
60
0uM FABP5 + 1.8uM ANS
0uM FABP5 + 1 uM ANS
20
0
430 450 470 490 510 530 550
Wavelength (nm)
Fluorescence Assay
Waiting Time
• Scan every 5 mins for a total of 20 mins
• Fluorescence values have the least variability around 15-20 mins
• More time for FABP5 and ANS to equilibrate? Mixing? Temperature?
Fluorescence Assay
Method
Purpose: to calculate Ki for different
drugs
• Add varying concentrations of a
lipophilic drug to outcompete FABP5
binding with ANS
• Test Controls
• Positive Control – Arachidonic Acid
• Vehicle Control – EtOH
• Drug Screening
• Calculate Ki
Drug Screening
• Method
• Purpose: qualitative assessment of FABP5-
drug binding
• Positive control: Arachidonic Acid
• fluorescence decreased by 48.28%
• Drugs have been shown to bind FABP5:
• Ibuprofen
• Rosiglitazone
• Pioglitazone
• Fenofibrate
• THC
• Retinoic Acid
• Drugs have been shown to bind other FABPs:
• Progesterone
• Midazolam
• Diclofenac
Vehicle Control
• Purpose: Test how solvent can change fluorescence EtOH vs. DMSO
• EtOH decreases fluorescence
• What concentration of ANS good for drug screening (dynamic range)
• still 1μM of FABP5
Fluorescence Displacement Assay