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

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【Required Reagents】

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

【Detection Protocol (Reference)】

Detection Steps	Protocol 1 (37°C Rapid Detection)	Protocol 2 (Room Temperature Detection)
​Step 1:	4μL Tag1-M1 +4μL Tag2-M2+ 6μL Acceptor Beads，Light-protected/Green light	4μL Tag1-M1 +4μL Tag2-M2+ 6μL Acceptor Beads，Light-protected/Green light
Incubation	​37°C with shaking for 20 minutes，Light-protected/Green light	​Room temperature incubation for 60 minutes,，Light-protected/Green light
​Step 2:	Add 6μL Donor Beads，Light-protected/Green light	Add 6μL Donor Beads，Light-protected/Green light
Incubation	​37°C with shaking for 10 minutes，Light-protected/Green light	​Room temperature incubation for 30 minutes，Light-protected/Green light
Reading​	Instrument reading	Instrument reading

【Performance Validation】

•Sample Preparation:

Biotinylated 3xFlag (Bio-3xFlag) was pre-diluted to 31μg/mL (10μnM) using Universal Buffer 3 as stock solution, then serially diluted as follows:

C5

ID	Final Concentration (nM)	Universal Buffer 1 Volume (μL)	High Concentration Addition Volume (μL)
C12	1.0E+03	210	90μL stock
C11	3.0E+02	210	90μL C12
C10	1.0E+02极简规则下完成剩余性能验证内容：p>	180	90μL 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
3.0E-01	210	90μL C6
C4	1.0E-01	180极简规则下完成剩余所有内容：	90μL C5
C3	3.0E-02	210	90μL C4
C2	1.0E-02	180	90μL C3
C1	0	180	/

•Detection Reagent Preparation:

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

•37°C Incubation Mode Results:

Maximum signal: 3549814

Minimum signal: 752

EC50= 1.648nM

•Room Temperature Incubation Mode Results:

Maximum signal: 1114403

Minimum signal: 355

EC50= 2.038nM

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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.