HAbs-HRP,GoatAnti-Rabbit IgG [SAG20010]

Product Description

This antibody is an affinity-purified Goat Anti-Rabbit Immunoglobulin G (IgG) antibody, conjugated to Horseradish Peroxidase (HRP). It is specific for the heavy and light chains (H+L) of rabbit IgG and will detect both native and denatured rabbit IgG. This conjugate is ideal for the detection of rabbit primary antibodies in a variety of immunoassays, including Western Blot (WB), Immunohistochemistry (IHC), and Enzyme-Linked Immunosorbent Assay (ELISA).

Key Features

1.High Sensitivity and Low Background: Provides a strong, specific signal with minimal non-specific binding under optimized conditions.

2.Broad Specificity: Recognizes the H+L chains of rabbit IgG, ensuring robust detection of a wide range of rabbit primary antibodies.

3.Ready-to-Use Conjugate: Supplied as an HRP conjugate, eliminating the need for secondary incubation steps with a separate HRP-labeled antibody.

4.High Purity: Affinity-purified to ensure high specificity and low cross-reactivity with immunoglobulins from other species.

5.Stable Formulation: Provided in a stabilizing buffer for extended shelf life.

Recommended Usage (For Reference Only)

This product is intended for research use only. Not for diagnostic or therapeutic use.

Important Note: The optimal working dilution for any application should be determined by the user through a titration experiment. The following are recommended starting points.

A. Western Blot (WB)

   Recommended Dilution: 1:1,000 to 1:4,000

   Protocol:

    1.  After protein transfer and blocking, incubate the membrane with the diluted primary antibody (rabbit).

    2.  Wash the membrane to remove unbound primary antibody.

    3.  Incubate the membrane with the Goat Anti-Rabbit IgG (HRP) conjugate, diluted in blocking buffer or TBST/ PBST.

    4.  Wash thoroughly to remove unbound conjugate.

    5.  Detect using a compatible HRP substrate (e.g., ECL, TMB) according to the substrate's manufacturer instructions.

B. Immunohistochemistry (IHC) - Paraffin-embedded Sections

   Recommended Dilution: 1:100 to 1:400

   Protocol:

    1.  Perform deparaffinization, rehydration, and antigen retrieval on tissue sections.

    2.  Block endogenous peroxidases and non-specific sites.

    3.  Incubate with the rabbit primary antibody.

    4.  Wash to remove unbound primary antibody.

    5.  Incubate with the Goat Anti-Rabbit IgG (HRP) conjugate, diluted in an appropriate buffer.

    6.  Wash thoroughly.

    7.  Develop the signal with a chromogenic substrate (e.g., DAB, AEC). Counterstain if desired.

C. ELISA

   Recommended Dilution: 1:2,000 to 1:8,000

   Protocol:

    1.  Coat the plate with antigen.

    2.  Block the plate to prevent non-specific binding.

    3.  Incubate with the rabbit primary antibody.

    4.  Wash to remove unbound primary antibody.

    5.  Incubate with the Goat Anti-Rabbit IgG (HRP) conjugate, diluted in blocking or assay buffer.

    6.  Wash thoroughly.

    7.  Add HRP substrate (e.g., TMB). Stop the reaction and measure the absorbance.

Precautions

Storage: Store at 4°C for frequent use (stable for at least 6 months). For long-term storage (>6 months), aliquot and store at -20°C. Avoid repeated freeze-thaw cycles, as this can degrade the HRP enzyme and antibody.

   Handling: Use gloves and practice standard laboratory safety procedures.

   Sodium Azide: This product contains sodium azide as a preservative. Azides can form explosive compounds with lead and copper plumbing. Dispose of waste with copious water rinsing to prevent azide buildup.

   Do Not Mix Lots: Do not mix different lots of the antibody.

   Optimization: The optimal dilution can vary depending on the specific experimental conditions (e.g., primary antibody concentration, incubation time, sample type). Always perform a pilot experiment to determine the best conditions for your system.

Frequently Asked Questions

Q1: What is the recommended storage condition?

   A: For short-term, store undiluted at 4°C. For long-term, aliquot and store at -20°C. Avoid freeze-thaw cycles.

Q2: Why is my signal too weak or absent?

   A: Possible reasons include:

       The antibody dilution is too high (too much dilution). Try a lower dilution (e.g., 1:500 for WB).

       The primary antibody is not binding effectively.

       The HRP substrate is inactive or has expired.

       Incubation times are too short.

       The membrane or sample contains residual contaminants that inhibit HRP.

Q3: Why is my background too high?

   A: Possible reasons include:

       The antibody dilution is too low (not enough dilution). Try a higher dilution (e.g., 1:10,000 for WB).

       Inadequate blocking or washing.

       The primary antibody concentration is too high.

       Over-development with the substrate. Reduce the development time.

Q4: Can this antibody be used for detecting native proteins?

   A: Yes. The H+L specificity allows it to bind to native rabbit IgG in techniques like IHC, ICC, and ELISA, as well as denatured IgG in Western Blot.

Q5: Does this antibody cross-react with serum from other species?

   A: It is affinity-purified against rabbit IgG to minimize cross-reactivity. However, always check the manufacturer's datasheet for specific cross-reactivity testing and include appropriate controls (e.g., no primary antibody) in your experiment to rule out non-specific binding.

Q6: What is the concentration of this antibody?

   A: Please refer to the Certificate of Analysis (CoA) provided with the specific product lot for the exact concentration.

Product Statement  

This product is intended for research use only. If used for the storage of valuable samples, please conduct small-scale testing prior to large-scale experiments. The product warranty is limited to the product itself and does not cover any other associated liabilities. Please be advised.

This product is for research use only and is not intended for clinical diagnosis or treatment.