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Technical Specification: Antibodies

1. Product Types

Solarbio antibodies are manufactured using hybridoma technology or recombinant expression and validated for specific research applications. Secondary antibodies are affinity-purified to minimize cross-reactivity. Isotype controls are matched to primary antibody species and subclass.

Type Format Available Hosts Available Conjugations
Primary antibodies — polyclonal Purified IgG, whole antiserum Rabbit, mouse, goat Unconjugated
Primary antibodies — monoclonal Purified IgG, ascites-free Mouse, rabbit Unconjugated, biotin
Secondary antibodies Affinity-purified F(ab′)₂ or whole IgG Goat anti-rabbit, goat anti-mouse, donkey anti-goat HRP, FITC, TRITC, Cy3, Cy5, Biotin
Control antibodies IgG isotype controls (matching subclass) Mouse IgG1, IgG2a, IgG2b; rabbit IgG Various
Antibody diluent Ready-to-use — —
Antibody stripping buffer Ready-to-use — —

2.1 Primary Antibody Dilutions

Application Starting Dilution Optimal Range Incubation Buffer
Western blot 1:500 1:500–1:2000 1 h at RT or overnight at 4°C 5% BSA or 5% non-fat milk in TBST
IHC (paraffin) 1:100 1:50–1:200 Overnight at 4°C PBS or Tris buffer
Immunofluorescence 1:200 1:100–1:500 1 h at RT or overnight at 4°C PBS + 1% BSA + 0.1% Triton X-100
Flow cytometry 1:100 1:50–1:200 30–60 min at 4°C (dark) FACS buffer (PBS + 1% BSA + 0.1% NaN₃)
ELISA 1:1000 1:500–1:5000 1 h at 37°C ELISA diluent (blocking buffer)
Immunoprecipitation 1:50 1:20–1:100 2 h at 4°C to overnight Low-stringency lysis buffer

2.2 Secondary Antibody Dilutions

Application Host – Target Conjugate Recommended Dilution Storage After Dilution
Western blot Goat anti-rabbit IgG HRP 1:5000–1:10000 Prepare fresh; discard after use
Western blot Goat anti-mouse IgG HRP 1:5000–1:10000 Prepare fresh
IHC (paraffin) Goat anti-rabbit IgG HRP 1:200–1:500 Prepare fresh
Immunofluorescence Goat anti-rabbit IgG FITC 1:500–1:1000 Use within 1 h; protect from light
Immunofluorescence Goat anti-mouse IgG Cy3 1:500–1:1000 Use within 1 h; protect from light
Flow cytometry Goat anti-mouse IgG FITC 1:200–1:500 Prepare fresh; protect from light
Flow cytometry Goat anti-rabbit IgG PE 1:200–1:500 Prepare fresh; protect from light
ELISA Goat anti-rabbit IgG HRP 1:5000–1:10000 Prepare fresh

3. Quality Control Specifications

Parameter Specification Test Method
Purity (SDS-PAGE) ≥95% for affinity-purified; ≥90% for whole IgG SDS-PAGE, Coomassie Blue staining
Antigen-specific ELISA titer >1:10,000 for polyclonal; EC₅₀ ≤ 100 ng/mL for monoclonal Indirect ELISA against immunogen
Endotoxin <1.0 EU/mg for in vivo-grade antibodies LAL chromogenic assay
BSA content <5% (BSA-free formulations available on request) Bradford assay
Aggregation (SEC-HPLC) <5% aggregates for purified IgG Size exclusion chromatography
Buffer PBS (pH 7.4) with 0.02% sodium azide —
Sterility Sterile-filtered (0.22 μm) USP <71>
Storage -20°C (aliquoted) or 2–8°C —
Shelf life 12 months at -20°C; 6 months at 2–8°C —

4. Conjugate Selection Guide

Conjugate Ex/Em (nm) Laser Line Application Relative Brightness
Unconjugated — — WB, IHC, IP, ELISA N/A
HRP 370/420 (TMB), 550/570 (DAB) — WB, IHC, ELISA High
Biotin — — Amplification systems (WB, IHC, ELISA) Very high (streptavidin amplification)
FITC 495/520 488 nm FACS, IF Medium
TRITC 557/576 543 nm IF, FACS Medium
Cy3 552/570 543 nm IF, FACS High
Cy5 649/670 633 nm FACS, multiplex IF High
PE (R-Phycoerythrin) 565/576 488 nm FACS Very high
APC (Allophycocyanin) 650/660 633 nm FACS High
PerCP 479/675 488 nm FACS (FL3) Medium

5. Antibody Validation Strategy

Solarbio antibodies are validated using a three-tier approach:

Validation Tier Method Acceptance Criterion
Tier 1 — Binding Direct ELISA against recombinant immunogen EC₅₀ ≤ 100 ng/mL (monoclonal); titer >1:10,000 (polyclonal)
Tier 2 — Specificity Western blot of cell lysates ± blocking peptide Single dominant band at predicted MW; band eliminated by peptide competition
Tier 3 — Application Application-specific testing Matches published MW in 2+ independent cell lines (WB); correct subcellular localization (IF)

5.1 Western Blot Validation (Example — Anti-β-Actin, Mouse Monoclonal)

Cell Lysate Expected MW Observed MW Specificity
HEK293 42 kDa 42 kDa Single band
HeLa 42 kDa 42 kDa Single band
A549 42 kDa 42 kDa Single band
Recombinant β-actin 42 kDa 42 kDa Single band

6. Storage and Handling

Formulation Short-term (≤3 months) Long-term (>3 months) Notes
Purified IgG (liquid) 2–8°C -20°C in aliquots Do not refreeze thawed aliquots
Purified IgG (lyophilized) 2–8°C -20°C after reconstitution Reconstitute at 1 mg/mL
Conjugated IgG (HRP) 2–8°C -20°C with 50% glycerol Store dark; avoid freeze-thaw
Conjugated IgG (FITC/Cy3/Cy5) 2–8°C (dark) -20°C in aliquots (dark) Protect from light
Whole antiserum 2–8°C -20°C 0.02% NaN₃ present
Isotype controls 2–8°C -20°C in aliquots Match to primary antibody

7. Troubleshooting

Issue Possible Cause Solution
High background (WB) Secondary antibody too concentrated; insufficient blocking Reduce secondary 1:10,000; increase blocking to 1 h at RT or overnight at 4°C
No signal (WB) Primary antibody expired; antigen degraded Verify antibody with positive control lysate; use fresh sample; add protease inhibitors
Multiple bands (WB) Non-specific binding; sample degradation Use blocking peptide competition; reduce gel loading; add fresh protease inhibitors
High background (IHC) DAB incubation too long; insufficient blocking Reduce DAB to 1–3 min; block with 10% normal serum for 30 min
Weak staining (IF) Primary antibody too dilute; fixation over-aggressive Titrate primary antibody 1:50; use 2–4% PFA instead of methanol fixation
Granular staining (IF) Antibody aggregates Centrifuge antibody at 12,000 × g for 10 min before use

8. Cross-References

For product procurement: solarbio.store