5-TAMRA-NHS

Guidelines (The following recommended experimental protocol is provided for guidance only and should be adjusted according to your specific requirements)

1. Stock Solution Preparation
1.1 Solvent: DMSO; Alternative solvent: DMF.
1.2 Concentration recommendation: High-concentration stock solution of 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: 0.1 M sodium phosphate buffer containing 0.01% Triton X-100 (adjust to the required pH); Alternative diluents: OptiMem medium supplemented with 10 mM HEPES, FRET buffer.
2.2 Working concentration: 1-30 nM for bacterial penicillin-binding protein assays; 0.03-333 nM for adherent cell assays.
2.3 Notes: Adjust the working solution concentration as needed; Prepare fresh immediately before use.

3. Staining Procedures
3.1 Sample Type Description
3.1.1 Applicable to purified soluble recombinant bacterial penicillin-binding proteins (PBPs)^[1]:
3.2 Incubation Conditions
3.2.1 Applicable to purified soluble bacterial PBPs^[1]: Incubate 1-30 nM 5-TAMRA-Ampicillin with the target PBP at ambient temperature for up to 40 min.
3.2.2 Applicable to adherent cells (HEK293T, HEK-293AD cells)^[2]: Incubate cells with 0.03-333 nM 5-TAMRA-labeled ligand at room temperature; Incubation with 50 nM ligand reaches binding saturation (within less than 1 minute); Incubation with other concentrations proceeds until binding equilibrium is achieved. Trypsin digestion is not required for live-cell imaging.
3.3 Washing Steps
3.3.1 Applicable to purified soluble bacterial PBPs: No specified washing step is required for fluorescence anisotropy/intensity assays.
3.3.2 Applicable to adherent cells: Wash cells with OptiMem medium supplemented with 10 mM HEPES or PBS buffer before imaging.

4. Control Setup
4.1 In bacterial PBP inhibition assays, a 0 μM inhibitor control (vehicle-only) is required to monitor the reaction between uninhibited PBPs and 5-TAMRA-Ampicillin.
4.2 In adherent cell assays, a displacement control is required: Add 50 μM SCH-23390 (1000-fold excess) to induce dissociation of the 5-TAMRA-labeled ligand from D1 receptors.

5. Detection and Analysis
5.1 Instrument types: Fluorescence microplate reader equipped with polarizing filter blocks; Laser scanning confocal microscope.
5.2 Excitation/emission wavelengths: 5-TAMRA-Ampicillin: 540 Ex/590 Em (microplate reader filter channels; intrinsic ~546/580 nm for 5-TAMRA-NHS ester); 5-TAMRA-labeled D1 ligand (UR-NR435): 561 Ex/569-669 Em detection (intrinsic 559/586 nm).
5.3 Result Analysis
5.3.1 Applicable to purified soluble bacterial PBPs:
5.3.1.1 Fluorescence anisotropy change: Upon binding to PBP, fluorescence anisotropy increases by approximately 110 milli-units (E. coli PBP2) or increases significantly (P. aeruginosa PBP2).
5.3.1.2 Fluorescence intensity change: Fluorescence intensity decreases significantly after ligand binding to P. aeruginosa PBP1a/PBP3, A. baumannii PBP1a/PBP3, and E. coli PBP3.
5.3.1.3 Kinetic analysis: Fit reaction progress curves to a competitive inhibition kinetic model via numerical integration to calculate the bimolecular rate constant k_inact/Ki for evaluating inhibitor potency.
5.3.2 Applicable to adherent cells:
5.3.2.1 Fluorescence intensity change: After adding 50 nM 5-TAMRA-labeled ligand, fluorescence intensity increases rapidly and reaches saturation within less than 1 minute; After adding the displacement control, fluorescence intensity decreases gradually.
5.3.2.2 Fluorescence localization: Fluorescence accumulates on the cell surface (consistent with the localization of D1 receptors).
5.3.2.3 Signal characteristics: 5-TAMRA produces a red/orange fluorescent signal.

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