HIR2 Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
The HIR2 antibody is a well-characterized monoclonal antibody targeting CD235a (Glycophorin A), widely used in erythroid cell research. Below are methodologically focused FAQs addressing key experimental considerations:
How to validate HIR2 antibody specificity in flow cytometry?
Three-step validation protocol:
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Positive controls: Use K562 (human erythroleukemia) and HEL (erythroid) cell lines showing ≥95% positivity by flow cytometry
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Negative controls: Test on CD235a-negative cells (e.g., Jurkat T-cells) with ≤2% background staining
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Competition assay: Pre-incubate with 10 µg/mL Glycophorin A peptide (N-terminal) to block >90% signal
Validation table:
| Parameter | Requirement | Typical Results |
|---|---|---|
| % Positivity (K562) | ≥90% | 95-98% |
| MFI Ratio | ≥10:1 | 15-20:1 |
| Lot Consistency | CV <15% | 8-12% |
What are HIR2's cross-reactivity limitations?
While primarily targeting Glycophorin A (GYPA), HIR2 shows:
Optimizing HIR2 for multi-color flow panels:
Critical parameters for panel design:
| Fluorophore | Compatible Lasers | Recommended Panel Position |
|---|---|---|
| FITC | 488 nm Blue | Channel 1 (FITC) |
| APC | 633-647 nm Red | Channel 5 (APC) |
| PE | 561 nm Yellow-Green | Channel 3 (PE) |
Pro Tip: When combining with CD71 (transferrin receptor), use HIR2-APC (Ex/Em 633/660 nm) with CD71-BV421 (Ex/Em 405/421 nm) to minimize spectral overlap .
Resolving data contradictions in erythroid maturation studies
Common pitfalls and solutions:
Titration in rare cell populations:
Modified protocol for <1% target cells:
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Enhanced staining: Increase antibody to 0.5 µg/test (5x standard)
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Reduced background: Add 2% mouse serum to block Fc receptors
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Acquisition: Collect ≥10^6 events using high-throughput mode
Performance metrics:
| Cell Frequency | Detection Threshold | Recommended Replicates |
|---|---|---|
| 0.1-1% | 95% CI | n=5 |
| <0.1% | Poisson statistics | n=10 |
