Rabbit anti-Human IgG Antibody;HRP conjugated

What is the principle behind Rabbit anti-Human IgG (HRP conjugated) antibodies and how do they function in detection systems?

Rabbit anti-Human IgG (HRP conjugated) antibodies are secondary antibodies generated by immunizing rabbits with human immunoglobulin G. These antibodies specifically recognize and bind to the constant region of human IgG molecules. The horseradish peroxidase (HRP) enzyme is covalently linked to the antibody through a modified Nakane and Kawaoi procedure, creating a detection system that can be visualized through various substrates.

In immunodetection systems, these antibodies function through a two-step process:

• The primary antibody (human IgG) binds to the target antigen

• The HRP-conjugated rabbit anti-human IgG binds to the primary antibody

• When substrate is added, the HRP enzyme catalyzes a reaction producing a detectable signal

This system provides signal amplification as multiple secondary antibodies can bind to a single primary antibody, enhancing detection sensitivity across various applications including Western blot, ELISA, and immunohistochemistry .

What are the optimal dilution ranges for different applications of Rabbit anti-Human IgG (HRP conjugated)?

The optimal dilution varies significantly based on the specific application, detection method, and the particular antibody product being used. Below is a comprehensive dilution guide based on application:

It is critical to note that these ranges are guidelines. As stated in multiple product documentation: "Optimal dilutions should be determined by each laboratory for each application" . Performing antibody titration experiments is essential for achieving optimal signal-to-noise ratios in your specific experimental system .

How should Rabbit anti-Human IgG (HRP conjugated) antibodies be stored to maintain optimal activity?

Proper storage is critical for maintaining antibody activity. Storage conditions vary based on the antibody's form (lyophilized or liquid):

For lyophilized antibodies:

• Store at 2-8°C in unopened vials

• After reconstitution, antibodies can be stored at 2-8°C for approximately 6 weeks

• For long-term storage after reconstitution:

  • Aliquot and freeze at -70°C or below

  • Alternatively, add equal volume of glycerol (ACS grade or better) for a final concentration of 50%, and store at -20°C

For liquid antibodies:

• Store at -20°C for up to one year

• Most liquid preparations contain 50% glycerol to prevent freezing at -20°C

• Avoid repeated freeze/thaw cycles by preparing working aliquots

• Some formulations contain preservatives like 0.03% Proclin-300 or 0.1% Kathon

Working dilutions should be prepared on the day of use for optimal results . Some products have demonstrated stability for at least 6 months from date of receipt when stored as recommended .

What are the most common cross-reactivity concerns with Rabbit anti-Human IgG (HRP conjugated) antibodies?

Cross-reactivity is a significant concern that can impact experimental specificity. Several products explicitly address this issue:

The most common cross-reactivity concerns include:

• Cross-reactivity with immunoglobulins from other species, particularly:

  • Mouse IgG

  • Rat IgG

  • Goat IgG

  • Rabbit IgG (self-reactivity)

Several manufacturers produce highly cross-adsorbed versions to minimize these concerns. For example:

• "This is a highly cross-adsorbed rabbit anti-human IgG (H+L) secondary antibody... To minimize cross-reactivity, the antibody has been adsorbed against mouse serum"

• "Based on Immunoelectrophoresis, no reactivity is observed to non-immunoglobulin human serum immunoglobulins"

• "This HRP conjugated antibody is specific for human IgG and shows no cross-reactivity with rat/mouse/goat/rabbit IgG"

When working with samples that may contain rheumatoid factor or other endogenous antibodies, additional blocking steps or specialized antibody fragments (F(ab')2) may be necessary to reduce non-specific binding .

What controls should be included when using Rabbit anti-Human IgG (HRP conjugated) in immunoassays?

Implementing appropriate controls is essential for result validation. Based on research methodologies described in the search results, the following controls should be included:

For Western Blot:

• Positive control: Known sample containing target protein with human IgG primary antibody

• Negative control: Omission of primary antibody

• Loading control: Housekeeping protein (e.g., beta-actin at 42kDa)

• Molecular weight marker: To verify target protein size

For Immunohistochemistry:

• Positive control: Tissue known to express the target protein

• Negative control: "Control was done by omitting primary antibody"

• Isotype control: Irrelevant human IgG of the same isotype

For ELISA:

• Standard curve: Serial dilutions of purified human IgG

• Blank wells: All reagents except primary antibody

• Cross-reactivity control: Testing with non-human immunoglobulins

As noted in one application example: "Detection of FoxP3 in human tonsil using 5 μg/mL of Rabbit monoclonal anti-human FoxP3 primary antibody (MAB8214) followed by 30 min incubation with Goat anti Rabbit HRP conjugated secondary antibodies... Control was done by omitting primary antibody" .

How does the HRP conjugation method affect antibody performance?

The HRP conjugation method significantly impacts antibody performance in terms of sensitivity, specificity, and stability:

Most manufacturers use a modified Nakane and Kawaoi procedure (J. Histochem. Cytochem. 1974, 22, 1084) for conjugating HRP to antibodies . This method creates a covalent bond between the enzyme and antibody while preserving both immunological and enzymatic activities.

Key factors affected by conjugation method:

• Conjugation ratio (HRP:antibody):

  • Higher ratios increase sensitivity but may reduce specificity

  • Excessive conjugation can sterically hinder antibody binding

• Site of conjugation:

  • Conjugation at or near the antigen-binding site can reduce affinity

  • Site-directed conjugation preserves binding capacity better than random conjugation

• Stability of conjugate:

  • Different conjugation chemistries yield varying stability profiles

  • Some conjugates require stabilizing additives like BSA (15mg/mL)

• Background signal:

  • Certain conjugation methods can increase non-specific binding

  • Purification steps post-conjugation remove free HRP that could contribute to background

For example, one product specifies: "Peroxidase conjugates are prepared by a modified Nakane and Kawaoi procedure" , while another indicates "HRP-labeled Rabbit IgG in phosphate buffered saline" , suggesting differences in conjugation approaches between manufacturers.

What troubleshooting approaches are most effective for high background when using Rabbit anti-Human IgG (HRP conjugated)?

High background is a common issue when using HRP-conjugated secondary antibodies. Based on the technical information provided in the search results, effective troubleshooting approaches include:

For Western Blot:

• Optimize antibody dilution:

  • Test serial dilutions (e.g., 1:5000, 1:10,000, 1:20,000) for ECL detection

  • Use higher dilutions for enhanced chemiluminescent detection systems

• Improve blocking:

  • Increase blocking time or concentration

  • Try alternative blocking agents (milk vs. BSA)

  • One product documents using "15mg/mL BSA" in their buffer formulation

• Adjust washing:

  • Increase number and duration of washes

  • Add 0.1-0.5% Tween-20 to wash buffer

  • Use "Immunoblot Buffer Group 1" as specified in some protocols

• Reduce cross-reactivity:

  • Use highly cross-adsorbed antibodies: "This is a highly cross-adsorbed rabbit anti-human IgG (H+L) secondary antibody... for specific staining with minimal background"

For Immunohistochemistry:

• Optimize antibody concentration:

  • Begin with recommended dilution (e.g., "1:20 dilution + DAB chromogen" )

  • Perform titration series to determine optimal concentration

• Modify incubation conditions:

  • Reduce incubation time (e.g., "30 min incubation" as used in documented protocols )

  • Perform incubations at 4°C overnight instead of room temperature

• Enhance blocking of endogenous peroxidase:

  • Pre-treat with hydrogen peroxide solution

  • Use specialized blocking reagents for endogenous biotin if using avidin-biotin systems

What factors influence the detection sensitivity when using Rabbit anti-Human IgG (HRP conjugated) with various substrates?

Detection sensitivity is influenced by multiple factors when using HRP-conjugated secondary antibodies with different substrates:

Substrate Characteristics:

Key Sensitivity Factors:

• Antibody concentration:

  • For ECL detection in Western blots: "1:10,000-1:20,000" dilution recommended for enhanced systems

  • For chromogenic detection: Higher concentrations typically required, "1:1000-1:3000"

• Incubation conditions:

  • Temperature affects enzyme kinetics

  • Time: "30 min incubation" specified for some IHC applications

• Buffer composition:

  • Presence of enzyme inhibitors

  • Metal ions: "Incompatible with sodium azide and metals incompatible with high phosphate concentrations"

• Substrate development time:

  • Excessive development increases background

  • Optimal timing must be determined empirically

• Detection instruments:

  • CCD imagers versus X-ray film for chemiluminescence

  • Spectrophotometer settings for ELISA applications

As noted in technical documentation: "Dilution factors are presented in the form of a range because the optimal dilution is a function of many factors, such as antigen density, permeability, etc. The actual dilution used must be determined empirically" .

How do different formulations of Rabbit anti-Human IgG (HRP conjugated) affect experimental outcomes?

Different formulations of Rabbit anti-Human IgG (HRP conjugated) can significantly impact experimental results through several mechanisms:

Buffer Components:

Various formulations exist across manufacturers:

• "0.01 M PBS (pH 7.4), 50% glycerol"

• "Phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 50% glycerol"

• "PBS (pH 7.1) with 15mg/mL BSA and 0.1% Kathon"

The presence of stabilizers like glycerol affects freezing properties and long-term stability, while preservatives like Proclin-300 or Kathon prevent microbial growth but may interfere with certain applications.

Antibody Fragments:

Different products offer varying antibody fragments:

• Whole IgG: "The whole IgG form of antibodies is suitable for the majority of immunodetection procedures and is the most cost effective"

• F(ab')2 fragments: "Antibody form: F(ab')2 fragment of affinity isolated antibody"

F(ab')2 fragments may reduce non-specific binding through Fc receptors, particularly important in tissues with high Fc receptor expression.

Purification Method:

• "Purified using the caprylic acid ammonium sulfate (CA-AS) precipitation method"

• "Affinity purified from rabbit antiserum"

The purification method affects specificity, with affinity purification generally yielding higher specificity antibodies.

Conjugation Ratio:

While specific conjugation ratios are rarely disclosed, different products may have varying HRP:antibody ratios, affecting signal intensity and non-specific binding.

The search results indicate that researchers should "determine optimal dilutions/concentrations for each specific application" to account for formulation differences.

What are the considerations for using Rabbit anti-Human IgG (HRP conjugated) in multiplexed assays?

Multiplexed assays require special considerations when using Rabbit anti-Human IgG (HRP conjugated) antibodies:

Cross-reactivity Management:

• Use highly cross-adsorbed antibodies: "This is a highly cross-adsorbed rabbit anti-human IgG (H+L) secondary antibody... To minimize cross-reactivity, the antibody has been adsorbed against mouse serum"

• Consider species compatibility: "This peroxidase conjugated antibody is specific for human IgG and shows no cross-reactivity with rat/mouse/goat/rabbit IgG"

Signal Separation Strategies:

When multiplexing with different detection systems:

• For dual HRP systems:

  • Sequential detection with peroxide inactivation between steps

  • Different substrates with distinct chromogenic products

• For combined HRP and other enzymes (like alkaline phosphatase):

  • Different substrates with non-overlapping detection wavelengths

  • Sequential detection protocols

Primary Antibody Selection:

• Host species considerations: "This human secondary antibody is ideal for investigators who routinely perform titration assays, western-blot, immunoprecipitation and more generally immunoassays"

• Isotype compatibility: Some antibodies detect specific human IgG isotypes or fragments

Technical Limitations:

• Signal overlap: HRP substrates may have broad emission spectra

• Sequential detection requirements

• Different optimal dilutions for each target: "Optimal dilutions should be determined by each laboratory for each application"

For example, a demonstrated application showed: "Detection of FoxP3 in human tonsil using 5 μg/mL of Rabbit monoclonal anti-human FoxP3 primary antibody (MAB8214) followed by 30 min incubation with Goat anti Rabbit HRP conjugated secondary antibodies (Catalog # HAF008) at 1:20 dilution + DAB chromogen (brown). The tissue was counterstained with Hematoxylin (blue)" . This demonstrates how HRP detection can be combined with counterstaining in a sequential protocol.

How does epitope specificity impact the performance of Rabbit anti-Human IgG (HRP conjugated) antibodies?

Epitope specificity is a critical factor determining the performance of Rabbit anti-Human IgG (HRP conjugated) antibodies:

Heavy and Light Chain Specificity:

Products vary in their epitope targeting:

• "(H+L)" specificity: "Rabbit Anti-Human IgG (H+L), Highly Cross-Adsorbed" - binds both heavy and light chains

• Heavy chain specific: "HRP Conjugated Rabbit Anti-human IgG (gamma-chain specific) secondary antibody"

• Fragment specific: Some antibodies target only Fc or Fab regions

This specificity affects:

• Cross-reactivity with other immunoglobulin classes

• Binding capacity per primary antibody molecule

• Potential interference from free light chains in samples

Clinical and Research Impact:

Epitope specificity influences detection in samples containing:

• Rheumatoid factor: Can bridge primary and secondary antibodies

• Free light chains: Present in certain disease states

• Immune complexes: May block epitopes

As noted in customer inquiries: "What exactly does Anti-IgG detect? Anti-IgG detects IgG" , but the specific epitopes recognized can vary between products.

Application-Specific Considerations:

For Western blotting applications like "PARP-cleavage upon treatment with embryonic stimuli" , using antibodies specific for the correct IgG fragment ensures accurate target identification.

For IHC applications like "Detection of FoxP3 in Human Tonsil" , epitope accessibility in fixed tissues may favor certain epitope specificities.

Understanding the epitope specificity is essential for selecting the appropriate antibody, particularly for specialized applications or when working with complex samples containing various immunoglobulin fragments.

What are the differences between polyclonal and monoclonal Rabbit anti-Human IgG (HRP conjugated) antibodies?

The search results indicate that most commercially available Rabbit anti-Human IgG (HRP conjugated) antibodies are polyclonal , but understanding the differences is important for research applications:

Polyclonal Rabbit anti-Human IgG:

• Production method: "The antiserum was developed in rabbit using the human IgG as the immunogen"

• Epitope coverage: Recognize multiple epitopes on human IgG

• Advantages:

  • Higher avidity due to recognition of multiple epitopes

  • More robust to minor sample variations

  • Often more sensitive for detection of low abundance targets

• Applications: "ELISA WB IHC"

Monoclonal Rabbit anti-Human IgG:

• Production method: Generated from single B-cell clones (less common in rabbit systems)

• Epitope coverage: Recognize a single epitope on human IgG

• Advantages:

  • Higher specificity for particular IgG epitopes

  • Greater lot-to-lot consistency

  • May have lower background in some applications

Performance Differences:

Some products emphasize their production methodology: "This antibody is purified from antiserum by immunoaffinity chromatography which removes essentially all rabbit serum proteins, except the specific antibody for human IgG" , indicating efforts to enhance the specificity of polyclonal preparations.

How do temperature and pH affect the activity and stability of Rabbit anti-Human IgG (HRP conjugated) antibodies?

Temperature and pH significantly impact both the HRP enzyme activity and antibody binding capacity:

Temperature Effects:

• Storage temperature recommendations:

  • Long-term storage: "-20°C for one year from date of receipt"

  • Working solutions: "2-8°C for approximately 6 weeks"

  • Freeze/thaw stability: "Avoid repeated freezing and thawing"

• Working temperature considerations:

  • Most immunodetection protocols specify room temperature incubations

  • Example protocol: "30 min incubation" at unspecified temperature

  • HRP enzyme activity increases with temperature, potentially enhancing sensitivity but also background

pH Effects:

• Buffer pH specifications:

  • Most formulations maintain pH 7.1-7.4:

    • "0.01 M PBS (pH 7.4)"

    • "pH 7.1 with 15mg/mL BSA"

• HRP enzyme activity:

  • Optimal pH range: 6.0-6.5 for maximum enzymatic activity

  • Substrate-dependent pH requirements

  • Performance declines rapidly outside pH 5-8 range

• Antibody binding:

  • Optimal binding typically occurs at physiological pH (7.2-7.4)

  • Extreme pH can permanently denature antibodies

Stability Considerations:

Buffer components enhance stability against temperature and pH variations:

• Glycerol (50%) prevents freezing damage and enhances stability

• BSA (15mg/mL) prevents adsorption to surfaces

• Preservatives (0.03% Proclin-300, 0.1% Kathon) prevent microbial growth

For optimal performance, researchers should follow manufacturer storage recommendations and avoid exposing antibodies to extreme conditions, as indicated by storage instructions across multiple products.

What methodologies exist for enhancing signal-to-noise ratio when using Rabbit anti-Human IgG (HRP conjugated) with low abundance targets?

Detecting low abundance targets requires specialized approaches to enhance signal-to-noise ratio:

Signal Amplification Strategies:

• Enhanced chemiluminescent systems:

  • Standard ECL: "SignalFire™ ECL Reagent #6883 1:1K–1:3K"

  • Enhanced systems: "SignalFire™ Elite ECL Reagent #12757 1:10K-1:20K"

  • Different sensitivity tiers available: "SignalFire™ Plus ECL Reagent #12630 1:5K-1:15K"

• Tyramide signal amplification (TSA):

  • Utilizes HRP to catalyze deposition of additional tyramide-HRP complexes

  • Can increase sensitivity 10-100 fold

Noise Reduction Approaches:

• Highly specific antibodies:

  • "This is a highly cross-adsorbed rabbit anti-human IgG (H+L) secondary antibody... To minimize cross-reactivity, the antibody has been adsorbed against mouse serum"

  • "Purified through immunoaffinity chromatography to remove all host serum proteins, except the specific antibody"

• Optimized blocking:

  • Buffer optimization: Some formulations include "15mg/mL BSA"

  • Extended blocking times

• Advanced washing techniques:

  • Increased number of washes

  • Extended wash durations

  • Addition of detergents or salt to reduce non-specific binding

Detection Optimization:

• Instrument sensitivity:

  • "Equipment Needed: WB/ELISA instrumentation; X-ray film cassette or a charge-coupled device (CCD) imager"

  • CCD imaging with extended exposure times for weak signals

• Substrate selection:

  • Enhanced chemiluminescent substrates for WB

  • High-sensitivity chromogenic substrates for IHC/ELISA

• Dilution optimization:

  • "The actual dilution used must be determined empirically"

  • Titration experiments to determine optimal concentration

These approaches are particularly relevant for applications such as detecting "cleavage (full length PARP 116kDa; cleaved PARP 89 kDa)" where both high and low abundance fragments must be visualized simultaneously.

What are the considerations for using Rabbit anti-Human IgG (HRP conjugated) in automation and high-throughput screening?

Implementation of Rabbit anti-Human IgG (HRP conjugated) antibodies in automated and high-throughput systems requires specific considerations:

Stability and Handling:

• Long-term stability at working dilution:

  • "Prepare fresh working dilution daily"

  • May require specialized stabilizers for robotic systems

  • Some formulations contain "0.03% Proclin-300" or "0.1% Kathon" as preservatives

• Temperature management:

  • "Store at -20°C" for stock solutions

  • Many liquid formulations contain "50% glycerol" to prevent freezing

  • Robotic systems may require room temperature stability

Protocol Standardization:

• Incubation time standardization:

  • Example: "30 min incubation" in manual protocols

  • May require optimization for automated liquid handlers

• Wash protocols:

  • Buffer composition standardization

  • Precision in wash volumes and timing

• Detection systems:

  • Compatible with plate readers, imaging systems

  • Substrate stability during automated runs

Lot-to-Lot Consistency:

• Quality control metrics:

  • "The sensitivity of each lot of antibody is confirmed using ELISA. The specificity of each lot of antibody is confirmed by immunoelectrophoresis (IEP)"

  • Critical for longitudinal high-throughput studies

• Validation procedures:

  • Standard curves with known concentrations

  • Internal controls for normalization

Technical Specifications:

• Compatible detection systems:

  • "Western blot(ECL): 0.1-0.3μg/ml"

  • "ELISA: 0.1-0.2μg/ml"

  • Optimal parameters must be established for specific automation platforms

• Buffer compatibility:

  • "Incompatible with sodium azide and metals incompatible with high phosphate concentrations"

  • Important consideration for automated liquid handlers

For high-throughput applications like screening, consistent antibody performance across large numbers of samples is essential, making standardized protocols and careful quality control particularly important.