Rat IgG2b Mouse mAb (HRP): Usage & Protocol Guide

Rat IgG2b Mouse mAb (HRP) is a horseradish peroxidase-conjugated mouse monoclonal antibody specifically raised against rat IgG2b, designed to enable sensitive and selective immunodetection of rat IgG2b-isotype primary antibodies across a range of standard immunoassay platforms.

Product Name

Rat IgG2b Mouse mAb (HRP)

SKU

Ab349244

Antibody Type

Mouse monoclonal antibody (secondary)

Conjugate

Horseradish Peroxidase (HRP)

Target Specificity

Rat IgG2b isotype

Reference Number

absea#KH-077

Product Overview

Rat IgG2b Mouse mAb (HRP) (SKU: Ab349244) is a mouse-derived monoclonal secondary antibody conjugated to horseradish peroxidase (HRP). It is specifically developed to recognize and bind the rat IgG2b immunoglobulin subclass, making it an essential reagent for isotype-specific immunodetection workflows. The HRP conjugate enables signal generation through standard chromogenic substrates such as TMB or DAB, or via chemiluminescent substrates such as ECL, allowing flexible integration into ELISA, Western blot (WB), immunohistochemistry (IHC), and related assay formats.

This antibody is particularly valuable in experimental contexts where isotype-specific secondary detection is required, for example when multiple rat IgG subclass antibodies are used simultaneously in multiplex or isotype-control experiments. The monoclonal nature of this reagent ensures batch-to-batch consistency and highly reproducible results.

Recommended Protocol

ELISA Protocol (Indirect):

1. Coat microplate wells with the antigen of interest at an appropriate concentration in carbonate-bicarbonate coating buffer (pH 9.6). Incubate overnight at 4 °C.
2. Wash wells three times with PBS containing 0.05% Tween-20 (PBST).
3. Block with 1–5% BSA or non-fat dry milk in PBS for 1 hour at room temperature.
4. Add the rat IgG2b primary antibody at the recommended dilution. Incubate for 1–2 hours at room temperature or overnight at 4 °C. Wash three times with PBST.
5. Add Rat IgG2b Mouse mAb (HRP) (Ab349244) diluted to the optimal working concentration in blocking buffer. Incubate for 1 hour at room temperature. Wash three to five times with PBST.
6. Add HRP substrate (e.g., TMB) and incubate for 10–30 minutes at room temperature, protected from light.
7. Stop the reaction with 2 M H₂SO₄ stop solution and read absorbance at 450 nm.

Western Blot Protocol:

1. Resolve proteins by SDS-PAGE and transfer to nitrocellulose or PVDF membrane.
2. Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature.
3. Incubate with rat IgG2b primary antibody overnight at 4 °C. Wash three times with TBST.
4. Incubate with Rat IgG2b Mouse mAb (HRP) (Ab349244) at the recommended dilution in blocking buffer for 1 hour at room temperature.
5. Wash three times with TBST and develop with ECL substrate. Image using X-ray film or digital imager.

Note: Optimal dilution for Ab349244 should be determined empirically for each application. Refer to the product datasheet for guidance.

Storage, Handling, and Stability

Proper storage and handling of HRP-conjugated antibodies are critical to maintaining enzymatic activity and immunoreactivity over time. The following guidelines are recommended for Rat IgG2b Mouse mAb (HRP) (Ab349244):

• Storage temperature: Store at 2–8 °C for short-term use. For long-term storage, aliquot and store at −20 °C. Avoid repeated freeze-thaw cycles, as these can denature the HRP enzyme and reduce activity.
• Light sensitivity: HRP activity can be reduced by prolonged exposure to light. Store the antibody in a light-protected environment.
• Sodium azide: If the formulation contains sodium azide, note that azide is a potent inhibitor of HRP. Ensure no azide contamination occurs during assay setup. Do not add sodium azide to working solutions containing this product.
• Protein concentration: Do not dilute the antibody until ready for use. Diluted antibody solutions should be prepared fresh and used promptly.
• Expiry: Refer to the product datasheet for specific expiry date information. Do not use reagents past their expiry date.

Troubleshooting

If unexpected results are observed when using Rat IgG2b Mouse mAb (HRP) (Ab349244), consult the following troubleshooting guide:

Problem	Possible Cause	Suggested Solution
No signal detected	Antibody concentration too low; HRP activity lost; substrate expired	Optimize antibody dilution; check storage conditions; use fresh substrate
High background	Insufficient blocking; secondary antibody concentration too high; cross-reactivity	Increase blocking time or concentration; reduce secondary antibody dilution; include appropriate isotype controls
Weak signal	Over-washing; suboptimal substrate incubation time; low primary antibody expression	Reduce wash stringency; extend substrate incubation; verify primary antibody binding
Non-specific bands (WB)	Cross-reactivity with non-target proteins; insufficient blocking	Optimize blocking conditions; verify species and isotype specificity of primary antibody

Frequently asked questions

What applications is Rat IgG2b Mouse mAb (HRP) (Ab349244) validated for?

Rat IgG2b Mouse mAb (HRP) is suitable for use as a secondary detection antibody in standard immunoassay applications including ELISA, Western blot, and immunohistochemistry. The specific validated applications for Ab349244 should be confirmed on the product datasheet, as performance may vary by platform.

Can Rat IgG2b Mouse mAb (HRP) detect other rat IgG subclasses?

No. This antibody is a monoclonal reagent raised specifically against the rat IgG2b subclass. It is not expected to cross-react with other rat IgG subclasses such as IgG1, IgG2a, or IgG2c. This isotype specificity is particularly useful for multiplex experiments requiring distinct subclass detection.

Why should sodium azide be avoided when using HRP-conjugated antibodies?

Sodium azide is a potent inhibitor of horseradish peroxidase (HRP) and even trace amounts can completely abolish enzymatic activity, leading to signal loss. Researchers should ensure that buffers, diluents, and any other reagents used alongside HRP-conjugated antibodies are free of sodium azide.

How should Ab349244 be stored to maintain activity?

Store Rat IgG2b Mouse mAb (HRP) at 2–8 °C for routine use and at −20 °C for long-term storage. Avoid repeated freeze-thaw cycles and protect from light to preserve both HRP enzymatic activity and antibody immunoreactivity.

What is the significance of using an isotype-specific secondary antibody?

Isotype-specific secondary antibodies such as Rat IgG2b Mouse mAb (HRP) allow researchers to selectively detect only the primary antibody of a defined isotype in a sample. This is critical in multiplex assays or isotype-control experiments where non-specific detection of other subclasses could compromise data interpretation.

References

1. Harlow E, Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1988.
2. Absea Biotechnology. Product Reference KH-077: Rat IgG2b Mouse Monoclonal Antibody (HRP). Absea, Beijing.

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