BTBCT

Guidelines (the following is our recommended protocol; this protocol is provided for guidance only and should be modified according to your specific needs).
1. Protein labeling with BTBCT
1.1 Dissolve BTBCT at a concentration of 10 mg/mL in dry ethanol.
1.2 Dissolve the target protein (e.g., BSA or streptavidin) in 0.1 M sodium carbonate buffer to the desired concentration, typically 1 mg/mL.
1.3 Gradually add the BTBCT solution to the protein solution while stirring.
1.4 Maintain stirring at room temperature for 2 hours.
1.5 Filter the mixture using an appropriate size filter (usually 0.2 μm).
1.6 If necessary, further purify the labeled protein via affinity chromatography to ensure that only functionalized protein is collected.
1.7 Dialyze the purified protein against a suitable storage buffer (commonly containing 0.05% NaN3).
1.8 Store at 4°C or freeze as needed.

2. Indirect Serum TSH TR-IFMA Procedure
2.1 Coating the Microplate: Add 30 μL of coating buffer containing 15 μg/mL of anti-TSH McAb-05 to each well. Incubate at room temperature for 24 hours. Wash twice with wash buffer.
2.2 Blocking: Add 40 μL of blocking buffer (typically containing BSA or another protein) and incubate at room temperature for 6 hours. Wash and air dry.
2.3 Adding Samples and Standards: Add 10 μL of TSH standard or serum sample to be tested to each well. Add 10 μL of a mixture containing biotinylated anti-TSH McAb-04 and McAb-03 at a concentration of about 30 ng/μL. Incubate with gentle shaking at room temperature for 1 hour.
2.4 Washing: Thoroughly wash the microplate with wash buffer.
Adding Signal Generation Reagent: Add 20 μL of TSH assay buffer containing streptavidin-BSA-BTBCT-Eu complex to each well. Gently shake and incubate for another 20 minutes.
2.5 Final Washing: Wash four times and rinse twice with distilled water.
2.6 Fluorescence Measurement: Measure the fluorescence intensity of each well using a time-resolved fluorometer.

3. Direct Serum T4 TRFIA Procedure
3.1 Microplate Preparation: Use a microplate coated with anti-T4 antibody, typically at a concentration of 10 μg/mL. Incubate the coated antibody at 4°C overnight.
3.2 Blocking Non-Specific Sites: Block the microplate with blocking buffer (usually containing 1% BSA) at room temperature for 1-2 hours.
3.3 Adding Samples and Label: Add a predetermined amount of the labeled T4-BSA-BTBCT-Eu complex along with the serum sample to be tested or T4 standard to each well.
3.4 Competition Reaction: Incubate the plate at room temperature for 1-2 hours.
3.5 Washing: Thoroughly wash the microplate with wash buffer.
3.6 Fluorescence Measurement: Measure the fluorescence signal of each well using a time-resolved fluorometer.

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