Rat IgG2c Mouse mAb (HRP): Protocol & Usage Guide

Rat IgG2c Mouse mAb (HRP) is a horseradish peroxidase-conjugated mouse monoclonal antibody specifically recognizing rat IgG2c heavy chain, designed to enable sensitive and specific detection of rat IgG2c in immunoassay applications including ELISA and Western blot.

Product Name

Rat IgG2c Mouse mAb (HRP)

SKU

Ab350678

Conjugate

Horseradish Peroxidase (HRP)

Host Species

Mouse (Monoclonal)

Target

Rat IgG2c (heavy chain)

Formulation

PBS only (as indicated by supplier reference absea#KH-078)

Recommended Applications

ELISA, Western Blot

Overview and Principle

Rat IgG2c Mouse mAb (HRP) (Ab350678) is a mouse monoclonal antibody raised against rat IgG2c and directly conjugated to horseradish peroxidase (HRP). As a secondary detection reagent, it binds specifically to the IgG2c subclass of rat immunoglobulins, enabling highly selective signal generation in enzyme-linked immunoassays.

HRP catalyzes the oxidation of chromogenic substrates such as TMB (3,3′,5,5′-tetramethylbenzidine) or chemiluminescent substrates such as ECL reagents, converting antibody-binding events into quantifiable colorimetric or luminescent signals. The monoclonal format ensures batch-to-batch consistency and minimizes cross-reactivity compared with polyclonal alternatives.

The product is formulated in PBS only (supplier reference absea#KH-078), making it compatible with standard immunoassay buffers without the need for additional desalting steps.

Materials Required

• Rat IgG2c Mouse mAb (HRP) — Ab350678 (Aladdin Scientific)
• Appropriate blocking buffer (e.g., 5% non-fat dry milk in TBST, or 1% BSA in PBS)
• Wash buffer (PBST: PBS + 0.05% Tween-20, or TBST)
• HRP substrate: TMB solution for ELISA; ECL reagent for Western blot
• Microplate reader (450 nm with 620 nm reference for TMB) or chemiluminescence imaging system
• ELISA plates or PVDF/nitrocellulose membranes (application-dependent)
• Primary antibody targeting antigen of interest (raised in rat, IgG2c subclass)
• Rat IgG2c isotype control (recommended for signal validation)

Step-by-Step Protocol

ELISA

1. Coat: Adsorb capture antibody or antigen onto the microplate at 4 °C overnight. Discard solution and pat dry.
2. Block: Add blocking buffer (200 µL/well) and incubate for 1 h at room temperature (RT). Wash 3× with PBST (200 µL/well, 3 min each).
3. Primary antibody: Add rat IgG2c primary antibody diluted in blocking buffer; incubate 1–2 h at RT or overnight at 4 °C. Wash 3× with PBST.
4. Secondary antibody (Ab350678): Dilute Rat IgG2c Mouse mAb (HRP) in blocking buffer (recommended starting dilution 1:5,000–1:10,000; optimize empirically). Add 100 µL/well and incubate 1 h at RT. Wash 5× with PBST.
5. Detection: Add 100 µL TMB substrate/well; incubate 10–15 min at RT in the dark. Stop reaction with 50 µL 2 N H₂SO₄. Read absorbance at 450 nm (reference 620 nm).

Western Blot

1. Transfer: After SDS-PAGE, transfer proteins to PVDF or nitrocellulose membrane.
2. Block: Incubate membrane in blocking buffer for 1 h at RT with gentle agitation.
3. Primary antibody: Incubate with rat IgG2c primary antibody overnight at 4 °C. Wash 3× with TBST (10 min each).
4. Secondary antibody (Ab350678): Dilute Rat IgG2c Mouse mAb (HRP) in blocking buffer (recommended starting dilution 1:5,000–1:10,000; optimize empirically). Incubate 1 h at RT. Wash 3× with TBST.
5. Detection: Apply ECL substrate; expose to film or image with a chemiluminescence system.

Storage and Handling

Store Rat IgG2c Mouse mAb (HRP) (Ab350678) according to the datasheet supplied with the product. As a general guideline for HRP-conjugated antibodies formulated in PBS:

• Short-term (up to 1 month): Store at 4 °C. Do not freeze repeatedly.
• Long-term: Aliquot and store at −20 °C. Add carrier protein (e.g., 0.1% BSA) if the antibody is highly dilute, to prevent adsorptive loss.
• Avoid: Repeated freeze-thaw cycles, which degrade HRP enzyme activity. Avoid exposure to sodium azide (NaN₃), which inhibits HRP.
• Light sensitivity: HRP conjugates should be protected from prolonged light exposure.

Note: Always refer to the specific lot datasheet for confirmed storage conditions.

Troubleshooting

Problem	Possible Cause	Suggested Remedy
No signal	Antibody dilution too high; HRP inactivated; primary antibody not rat IgG2c subclass	Titrate secondary antibody; check storage conditions; confirm primary antibody subclass
High background	Insufficient blocking; secondary antibody concentration too high; inadequate washing	Optimize blocking buffer and duration; reduce secondary antibody concentration; increase wash steps
Weak signal	HRP partially inactivated by azide or freeze-thaw; substrate incubation too short	Use azide-free buffers; minimize freeze-thaw cycles; extend substrate incubation
Non-specific bands (WB)	Cross-reactivity with other rat IgG subclasses; membrane over-dried	Confirm subclass specificity; keep membrane moist during protocol

Frequently asked questions

What species and immunoglobulin subclass does Ab350678 detect?

Ab350678 is a mouse monoclonal antibody that specifically detects rat IgG2c. It will not reliably detect other rat IgG subclasses (e.g., IgG1, IgG2a, IgG2b) or immunoglobulins from other species without prior cross-reactivity validation.

Can this antibody be used with sodium azide-containing buffers?

No. Sodium azide (NaN₃) is a potent inhibitor of horseradish peroxidase (HRP) and must not be present in the working dilution buffer or storage buffer if enzymatic activity is required. Ensure all reagents used with Ab350678 are azide-free during the detection step.

What is the recommended working dilution for Ab350678?

A starting dilution of 1:5,000 to 1:10,000 is typically appropriate for HRP-conjugated secondary antibodies in ELISA and Western blot; however, the optimal dilution should be determined empirically for each experimental system and antigen concentration. Always titrate the antibody in your specific assay conditions.

How should Ab350678 be stored to maintain HRP activity?

Store the antibody at 4 °C for short-term use (up to one month) and at −20 °C for long-term storage in single-use aliquots to avoid repeated freeze-thaw cycles. Keep the product protected from light and away from azide-containing solutions.

Is Ab350678 suitable for immunohistochemistry (IHC) or flow cytometry?

The source material specifies ELISA and Western blot as the primary recommended applications. Suitability for IHC or flow cytometry has not been confirmed in the available product information and would require independent validation. Please consult the product datasheet or contact Aladdin Scientific technical support for guidance.

References

1. Temporarily unavailable — no specific published reference was provided in the source material for Ab350678. Users should consult the product datasheet supplied with each lot.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.