Mouse Monoclonal Antibody Screening Set (His-tag Antigen)
Mouse Monoclonal Antibody Screening Set (His-tag Antigen)
Product Details
Product Details
Product Specification
| Stability & Storage | Store at 2～8°C away from light; product shelf life is 12 months. |
Background
Homogeneous Immuno Chemiluminescence Assay (HICA) is a homogeneous immunoassay method based on energy transfer and luminescence between donor beads and acceptor beads at close proximity.
Donor beads recognize Protein 1 (Tag1 label), while 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 the protein interaction.
This product features a simple operation process, no washing steps, fast speed, and high sensitivity, enabling the detection of weak interactions.
Components
Name |
Main Component |
2000T |
Reagent A |
Anti-His-tag antibody-crosslinked acceptor Beads |
10mL |
Reagent B |
Biotin-labeled anti-mouse IgG antibody |
5mL |
Reagent D |
Streptavidin Donor Beads |
5mL |
Protocol
【Required Reagents】
Name |
Catalog Number |
Universal Buffer 4 |
UA086115 |
[Testing Procedure for Reference]
Testing Step |
Procedure |
Step 1: |
Add His-tag antigen to Reagent A at a concentration of 2μg/mL |
Step 2: |
Mix Reagent A, Reagent B, and Reagent D in a volume ratio of 2:1:1 uniformly,avoid light/green light |
Step 3: |
Add 10μL of the sample to be tested + 10μL of the mixed reagent per test,avoid light/green light |
Incubation |
Shake and incubate at 37℃ for 60 minutes,avoid light/green light |
Reading |
Instrument reading |
【Sample and Reagent Preparation】
•Pre-dilute the M1 antibody to 250 ng/mL (1.67 nM) using Universal Buffer 4 as C12, then perform gradient dilution according to the following protocol:
ID |
Concentration (ng/mL) |
Universal Buffer 4 Volume (μL) |
High Concentration Addition Volume (μL) |
C12 |
2.50E+02 |
/ |
/ |
C11 |
7.50E+01 |
210 |
90μL C12 |
C10 |
2.50E+01 |
180 |
90μL C11 |
C9 |
7.50E+00 |
210 |
90μL C10 |
C8 |
2.50E+00 |
180 |
90μL C9 |
C7 |
7.50E-01 |
210 |
90μL C8 |
C6 |
2.50E-01 |
180 |
90μL C7 |
C5 |
7.50E-02 |
210 |
90μL C6 |
C4 |
2.50E-02 |
180 |
90μL C5 |
C3 |
7.50E-03 |
210 |
90μL C4 |
C2 |
2.50E-03 |
180 |
90μL C3 |
C1 |
0 |
180 |
/ |
【Performance Verification】
•Reaction curve:
Maximum signal: 1114027;
Minimum signal: 451
EC50= 60.05 ng/mL (0.40nM)
•Limit of blank:
C1 was tested 20 times repeatedly to calculate the mean signal and SD. The concentration value corresponding to the mean signal + 2×SD was calculated using the reaction curve, which is the limit of blank.
Limit of blank= 0.023ng/mL (0.15pM)
•Repeatability CV%:
High and low concentration samples were tested 10 times repeatedly to calculate the concentration CV%.
|
Low concentration |
High concentration |
Repeatability CV% |
4.64% |
2.19% |
•Specificity: Dilute the following proteins to 1μg/mL using Universal Buffer 4 and test the cross-reactivity rate.
Test Substance |
Cross-Reactivity Rate |
Human IgG |
0.00% |
Rabbit IgG |
0.00% |
Guidelines
1. This experiment is light-sensitive. Perform all procedures (preparation, pipetting, and incubation) in a green light environment (illuminance below 100 LUX) to avoid light exposure.
2. This product is compatible with multifunctional microplate readers equipped with an Alpha detection module.
3. To ensure comparability of experimental data across different batches, strictly control incubation temperature and duration.
4. Avoid bubble formation during pipetting.
