Anti-Flag Tag Acceptor Beads

Homogeneous Immuno Chemiluminescence Assay (HICA) is a homogeneous immunoassay method based on energy transfer between donor beads and acceptor beads for luminescence.

Donor beads recognize Protein 1 (Tag1 label), and Acceptor beads recognize Protein 2 (Tag2 label). When Protein 1 binds to Protein 2, the distance between the beads becomes less than 200nm. Upon excitation at 680nm, the donor beads generate singlet oxygen, which diffuses to the acceptor beads. The acceptor beads then undergo a redox reaction, emitting light at 615nm. The signal intensity is directly proportional to the strength of protein interaction.

This product features a simple operation process, requires no washing, and offers high speed and sensitivity. It can detect weak binding.

Specification	Fill Volume
250 μg	50 μL
5 mg	1 mL
25 mg	1 mL x 5

[Reagents Required]

Name	Catalog Number
Anti-Flag Tag Acceptor Beads	UA086103
Streptavidin Donor Beads	UA086104
Universal Buffer 3	UA086114

 

[Reference for the Assay Procedure]

Assay procedure	Assay procedure 1 (37℃ rapid assay)	Assay procedure 2 (room temperature assay)
​Step one:	4 μL Tag1-M1 + 4 μL Tag2-M2 + 6 μL Acceptor Beads,Protect from light/green light	4 μL Tag1-M1 + 4 μL Tag2-M2 + 6 μL Acceptor Beads,Protect from light/green light
Incubation	37℃ shaking incubation for 20 minutes,Protect from light/green light	Room temperature incubation for 60 minutes,Protect from light/green light
​Step two:	Add 6 μL Donor Beads,Protect from light/green light	Add 6 μL Donor Beads,Protect from light/green light
Incubate	Shake and incubate at 37°C for 10 minutes,Protect from light/green light	Incubate at room temperature for 30 minutes,Protect from light/green light
Read the results​	Instrument reading	Instrument reading

 

[Performance Verification]

•Sample Preparation:

Using Universal Buffer 3, pre-dilute biotinylated 3xFlag (Bio-3xFlag) to 31 μg/mL (10 μM) as the stock solution, then perform gradient dilutions according to the following scheme:

Number	Final Concentration (nM)	Universal Buffer 1 Volume (μL)	High-Concentration Addition Volume (μL)
C12	1.0E+03	210	90 μL of stock solution
C11	3.0E+02	210	90 μL of C12
C10	1.0E+02	180	90 μL of C11
C9	3.0E-01	210	90μL C10
C8	1.0E-01	180	90μL C9
C7	3.0E-00	210	90μL C8
C6	1.0E-00	180	90μL C7
C5	3.0E-01	210	90μL C6
C4	1.0E-01	180	90μL C5
C3	3.0E-02	210	90μL C4
C2	1.0E-02	180	90μL C3
C1	0	180	/

 

•Preparation of test reagents:

Name	Preparation concentration	Diluent
Anti-Flag Tag Acceptor Beads	25 μg/mL	Universal Buffer 1
Streptavidin Donor Beads	25 μg/mL	Universal Buffer 1

 

 

•37℃ incubation mode results:

 

Highest signal: 3,549,814

Lowest signal: 752

EC50 = 1.648 nM

•Room temperature incubation mode results:

Highest signal: 1,114,403

Lowest signal: 355

EC50 = 2.038 nM

1. This experiment is light-sensitive; ensure all procedures are performed under light-protected conditions. It is recommended to conduct preparation, sample loading, and incubation steps under green light (illuminance below 100 LUX). 2. This product is compatible with multifunctional microplate readers equipped with Alpha detection modules. 3. Vortex thoroughly before use or briefly centrifuge (2000×g, 5–10 seconds) to ensure complete sample retrieval. 4. It is advised to use the accompanying dilution buffer from our company for reagent preparation and sample dilution. If additional components are required, they may be directly added to this buffer. 5. To ensure comparability of experimental data across different batches, strictly control incubation temperature and duration. 6. Avoid bubble formation during sample loading.