Rabbit anti-Guinea Pig IgG Antibody

What is Rabbit anti-Guinea Pig IgG antibody and how is it produced?

Rabbit anti-Guinea Pig IgG antibody is a secondary antibody generated by immunizing rabbits with purified guinea pig immunoglobulin G (IgG). The production process typically involves several key steps:

• Immunization: Rabbits are immunized with guinea pig IgG whole molecule as the immunogen

• Antiserum collection: Antiserum is harvested from the rabbits after they have mounted an immune response

• Purification: The antibodies are purified using immunoaffinity chromatography, where guinea pig IgG is coupled to agarose beads

• Adsorption: Optional additional purification through solid phase adsorption to remove unwanted reactivities against other species

The resulting polyclonal antibody preparation recognizes epitopes on both the heavy and light chains of guinea pig IgG. Immunoelectrophoresis testing typically confirms specificity by showing a single precipitin arc against guinea pig IgG and guinea pig serum .

What does "H&L" or "Heavy & Light Chain" specificity mean for these antibodies?

The designation "H&L" or "Heavy & Light Chain" refers to the binding specificity of the antibody:

• Heavy Chain specificity: The antibody recognizes epitopes on the heavy chains of guinea pig IgG, which contain the Fc region and part of the Fab region

• Light Chain specificity: The antibody binds to epitopes on the light chains of guinea pig IgG

• H&L specificity: The antibody can bind to epitopes on both the heavy and light chains of guinea pig IgG

This is important because antibodies with H&L specificity will react with heavy chains of guinea pig IgG and with light chains of most guinea pig immunoglobulins, as stated in product specifications . This broader reactivity makes H&L antibodies versatile for most applications where detection of guinea pig IgG is required.

What applications can Rabbit anti-Guinea Pig IgG antibodies be used for?

Rabbit anti-Guinea Pig IgG antibodies have broad utility across multiple laboratory techniques. Based on validated protocols, these antibodies can be applied to:

These applications depend on the specific conjugation of the antibody and the experimental design requirements .

How do different conjugates affect the applications of Rabbit anti-Guinea Pig IgG antibodies?

The conjugate attached to a Rabbit anti-Guinea Pig IgG antibody determines its detection method and optimal applications:

• Enzyme conjugates (HRP): Ideal for colorimetric detection in Western blots, ELISA, and IHC applications. Provides amplified signal through enzymatic reaction with appropriate substrates. Offers high sensitivity but requires substrate addition and typically provides stable signal .

• Fluorescent conjugates:

  • TRITC/Rhodamine: Excitation ~550nm, emission ~570nm (red fluorescence). Used in fluorescence microscopy, FLISA, and flow cytometry .

  • DyLight 488: Excitation ~493nm, emission ~518nm (green fluorescence). Superior photostability compared to traditional fluorophores. Matches output wavelengths of common fluorescence instrumentation .

  • Cy3/Cy5/Cy5.5: Various excitation/emission profiles for multiplex applications. Particularly useful for multi-color imaging experiments .

• Non-conjugated antibodies: Used as bridging antibodies in specialized applications or when custom conjugation is preferred. Provides flexibility but requires additional detection steps .

The choice of conjugate should align with available detection instruments, desired sensitivity, and the need for multiplexing capabilities in the experimental design.

What are the proper handling and storage conditions for Rabbit anti-Guinea Pig IgG antibodies?

Proper handling and storage is critical for maintaining antibody functionality. The following guidelines are based on manufacturer recommendations:

For lyophilized antibodies:

• Store unopened at 2-8°C until reconstitution

• Reconstitute with deionized water (typically 1.0-1.1 mL)

• Allow to stand 30 minutes at room temperature to dissolve completely

• Centrifuge to remove any particulates

For reconstituted antibodies:

• For short-term storage (≤1 week): Store at 4°C

• For long-term storage: Dilute with 50% glycerol and store at -20°C as a liquid

• Avoid repeated freeze/thaw cycles which can denature the antibody

Working solutions:

• Prepare fresh working dilutions daily for optimal results

• Discard any unused diluted antibody after experiment completion

Stability testing indicates properly stored antibodies typically maintain activity for 12 months from date of receipt when following these guidelines .

How does pre-adsorption affect specificity, and when is it critical in experimental design?

Pre-adsorption (or "preadsorbed" antibodies) is a purification technique that significantly impacts antibody specificity by removing potential cross-reactivity with non-target species.

Mechanism of Pre-adsorption:
Pre-adsorption involves solid-phase adsorption where the antibody preparation is passed through a column containing immobilized proteins from species that might cross-react with the antibody. This process removes antibodies that bind to epitopes shared between guinea pig IgG and other species .

Specificity Profile Comparison:

When Pre-adsorption is Critical:

• Co-localization studies involving multiple species-derived antibodies

• Tissue samples containing endogenous IgG from multiple species

• When working with evolutionarily related species with high protein homology

• When background/non-specific signal compromises data interpretation

Immunoelectrophoresis testing of pre-adsorbed antibodies typically confirms a single precipitin arc against guinea pig IgG and guinea pig serum, with no reaction against the adsorbed species, validating the specificity enhancement .

What validation methods ensure specificity of Rabbit anti-Guinea Pig IgG antibodies?

Comprehensive validation is essential to confirm antibody specificity. Current best practices include multiple complementary approaches:

Standard Validation Techniques:

• Immunoelectrophoresis (IEP): This technique confirms specificity by demonstrating a single precipitin arc against:

  • Anti-Rabbit Serum (confirms antibody species)

  • Guinea Pig IgG (confirms target binding)

  • Guinea Pig Serum (confirms native protein recognition)

• ELISA Titer Testing: Determines sensitivity by measuring:

  • Positive titer against target antigen (Guinea Pig IgG)

  • Negative/minimal titer against potential cross-reactive species

• Western Blot Analysis: Confirms molecular weight specificity by demonstrating:

  • Binding to guinea pig IgG heavy chains (~50 kDa) and/or light chains (~25 kDa)

  • Absence of bands when testing cross-reactive species proteins

Advanced Validation Methods:

• CUT&RUN/CUT&Tag Validation: As demonstrated in validation study #104622, where:

  • Guinea pig anti-rabbit IgG antibody successfully increased protein A binding sites

  • Testing in human primary thymocytes and PER-117 cells confirmed specificity

  • Resulted in quantifiable amounts of tagmented genomic fragments

• Multiplexed Immunofluorescence: Demonstrates antibody specificity in complex samples by:

  • Co-staining with multiple antibodies against different targets

  • Confirming expected localization patterns

  • Verifying absence of overlap with non-target proteins

Validation experiments should include both positive controls (containing guinea pig IgG) and negative controls (lacking guinea pig IgG but containing potential cross-reactive species) to conclusively demonstrate specificity.

How can Rabbit anti-Guinea Pig IgG antibodies be optimized for CUT&RUN and CUT&Tag protocols?

CUT&RUN (Cleavage Under Targets and Release Using Nuclease) and CUT&Tag (Cleavage Under Targets and Tagmentation) are cutting-edge chromatin profiling techniques that require specific optimization of secondary antibodies like Rabbit anti-Guinea Pig IgG.

Optimization Parameters for CUT&RUN:

• Concentration Optimization:

  • Recommended working dilution: 1:100 (standard protocol)

  • A final concentration of approximately 1.2 mg/mL has been validated for optimal results

  • Higher concentrations may increase background without improving sensitivity

• Binding Conditions:

  • Incubation time: 30 minutes at 4°C after primary antibody binding

  • Wash buffer composition: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, with protease inhibitors

  • Multiple wash steps (typically 5-6) are critical to reduce background

CUT&Tag Protocol Adaptations:

Based on validation study #104174 from Tom Taghon's lab:

• When using rabbit monoclonal primary antibodies (such as anti-H3K27me3):

  • Primary antibody dilution: 1:100

  • Secondary guinea pig anti-rabbit antibody dilution: 1:100

  • pA-Tn5 fusion protein dilution: 1:1000

• Cell preparation considerations:

  • Protocol works efficiently with as few as 50,000 cells

  • Demonstrated successful implementation in both cell lines (PER-117) and primary cells (human thymocytes)

• Quality control metrics:

  • Successful optimization produces a ladder-like distribution pattern in fragment analysis

  • Library concentrations after PCR amplification should reach detectable levels (typically >0.1 ng/μL)

  • Negative controls using normal rabbit IgG should show minimal amplification

These optimizations have been validated to significantly increase the number of protein A binding sites for each bound rabbit primary antibody, enhancing the efficiency of chromatin capture in both techniques.

What critical parameters optimize signal-to-noise ratio in fluorescence applications?

Achieving optimal signal-to-noise ratios with fluorescently conjugated Rabbit anti-Guinea Pig IgG antibodies requires careful attention to several parameters:

Conjugate Selection Considerations:

Protocol Optimization Strategies:

• Antibody Titration:

  • Flow Cytometry: 1:500-1:2,500 dilution range

  • Fluorescence Microscopy: 1:1,000-1:5,000 dilution range

  • Always perform titration experiments to determine optimal concentration for specific samples

• Blocking Optimization:

  • Include 10 mg/mL BSA (immunoglobulin and protease-free) in formulation to reduce non-specific binding

  • For tissues with high endogenous IgG, use additional blocking with unlabeled secondary antibodies

• Washing Protocols:

  • Multiple (5-6) washes with 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2 buffer

  • Include 0.05-0.1% detergent (Tween-20 or Triton X-100) in wash buffer to reduce hydrophobic interactions

  • Extend final wash times to ensure complete removal of unbound antibody

• Sample Preparation Considerations:

  • Fixation method significantly impacts epitope accessibility and autofluorescence

  • For tissues with high autofluorescence, consider using Sudan Black B treatment

  • Photobleaching samples prior to antibody application can reduce background

• Imaging Parameters:

  • Use spectral unmixing for multiplex experiments to accurately separate fluorophore signals

  • Consider time-gated detection to separate antibody signal from short-lived autofluorescence

Implementation of these parameters has been validated to provide optimal signal-to-noise ratios across different fluorescence-based applications using Rabbit anti-Guinea Pig IgG antibodies.

How do you troubleshoot non-specific binding or weak signals in immunodetection methods?

Troubleshooting Rabbit anti-Guinea Pig IgG antibody performance requires systematic evaluation of multiple parameters:

Problem 1: High Background/Non-specific Binding

Problem 2: Weak or Absent Signal

Problem 3: Inconsistent Results

For persistent issues, analytical validation using immunoelectrophoresis is recommended, as this technique can "result in a single precipitin arc against anti-Rabbit Serum, Guinea Pig IgG and Guinea Pig Serum" confirming antibody specificity and functionality.

How can Rabbit anti-Guinea Pig IgG antibodies be utilized in multiplex immunoassays?

Multiplex immunoassays using Rabbit anti-Guinea Pig IgG antibodies require careful planning to avoid cross-reactivity while maximizing information obtained from a single experiment:

Strategic Design Considerations:

• Conjugate Selection for Spectral Separation:
  Selecting non-overlapping fluorophores is critical for clear signal discrimination:

  Conjugate	Excitation/Emission	Recommended Pairing With
  DyLight 488	493/518 nm	Cy5 (650/670 nm) or TRITC (550/570 nm)
  TRITC	550/570 nm	DyLight 488 (493/518 nm) or Cy5 (650/670 nm)
  Cy5	650/670 nm	DyLight 488 (493/518 nm) or TRITC (550/570 nm)
  DyLight 594	593/618 nm	DyLight 488 (493/518 nm) or Cy5.5 (675/694 nm)

• Host Species Combinations:
  When using multiple primary antibodies, choose combinations that avoid secondary antibody cross-reactivity:

  • Guinea pig primary + goat anti-guinea pig secondary

  • Rabbit primary + guinea pig anti-rabbit secondary

  This approach minimizes cross-reactivity as validated in studies: "This product was prepared from monospecific antiserum by immunoaffinity chromatography using Guinea Pig IgG coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities" .

Optimized Multiplex Protocol:

• Sequential Detection Method:

  • Apply first primary antibody → wash → apply first secondary antibody

  • Block with excess unconjugated IgG from host of first secondary

  • Apply second primary antibody → wash → apply second secondary antibody

• Validated Dilution Ratios:
  For multicolor imaging utilizing various commercial platforms:

  • Flow Cytometry: 1:500-1:2,500

  • Fluorescence Microscopy: 1:1,000-1:5,000

• Controls for Multiplex Validation:

  • Single primary-secondary pairs to establish baseline signal

  • Secondary-only controls to assess non-specific binding

  • Isotype controls to confirm specificity

These approaches have been validated to provide "multiplex analysis, including multicolor imaging, utilizing various commercial platforms" with minimal cross-reactivity between detection channels.

What considerations are important when using these antibodies in epigenetic research?

Rabbit anti-Guinea Pig IgG antibodies have become instrumental in advanced epigenetic research techniques, particularly for CUT&RUN and CUT&Tag methods that map protein-DNA interactions and chromatin states:

Key Experimental Design Factors:

• Antibody Quality Parameters:

  • Specificity: Pre-adsorbed antibodies minimize background in chromatin studies

  • Concentration: Standardized at 1.2 mg/mL for optimal results in CUT&RUN/CUT&Tag protocols

  • Purification: Immunoaffinity chromatography purification ensures minimal non-specific interactions

• Validated Epigenetic Applications:
  Based on published research using these antibodies:

  Application	Primary Antibody	Recommended Secondary	Cell Types Validated	Key Findings
  CUT&Tag	Rabbit anti-H3K27me3	Guinea pig anti-rabbit IgG	Human primary thymocytes, PER-117 cells	Successful profiling of H3K27me3 signals with ladder-like fragment distribution
  CUT&RUN	Rabbit IgG (negative control)	Guinea pig anti-rabbit IgG	HEK293T cells	Effective negative control showing minimal background

• Protocol-Specific Adaptations:

  • Cell number optimization: Protocols work with as few as 50,000 cells

  • Binding conditions: 1:100 dilution of both primary and secondary antibodies

  • MNase digestion parameters: Precise timing affects fragment size distribution

Quality Control Metrics:

For epigenetic applications, several quality indicators have been established:

• Library concentration after PCR amplification:

  • Positive controls (e.g., H3K27me3): 0.122-0.538 ng/μL

  • Negative controls (normal IgG): Below detection limit

• Fragment size distribution:

  • Ladder-like distribution pattern for histone modifications like H3K27me3

  • Lower cell numbers may show increased larger fragments

These parameters are critical when using Rabbit anti-Guinea Pig IgG antibodies in epigenetic research, as demonstrated in validation studies by both Tom Taghon's lab and the Cantù Lab .

How does antibody affinity purification impact experimental outcomes?

The purification method used for Rabbit anti-Guinea Pig IgG antibodies significantly impacts specificity, sensitivity, and reproducibility in experimental applications:

Purification Methods Comparison:

Performance Metrics by Purification Method:

• Immunoaffinity-Purified Antibodies:

  • Specificity: "Assay by immunoelectrophoresis resulted in a single precipitin arc against anti-Rabbit Serum, Guinea Pig IgG and Guinea Pig Serum"

  • Background: Minimal non-specific binding in negative control tissues

  • Dilution Range: Can be used at higher dilutions (1:20,000-1:100,000 for ELISA)

• Antibodies with Additional Pre-adsorption:

  • Cross-reactivity: "No reaction was observed against Bovine, Chicken, Goat, Hamster, Horse, Human, Mouse, Rabbit, Rat or Sheep Serum Proteins"

  • Application Versatility: Essential for multiplex imaging and complex tissue analysis

  • Background: Further reduced background in tissues containing multiple species proteins

Impact on Experimental Reproducibility:

The purification method directly affects experiment-to-experiment consistency:

• Batch consistency: Immunoaffinity purification provides more consistent lot-to-lot performance

• Protocol robustness: Highly purified antibodies require less protocol optimization between experiments

• Data quality: Cleaner signals result in more reproducible quantitative analysis

When selecting Rabbit anti-Guinea Pig IgG antibodies, researchers should consider the purification method based on their specific application requirements, with immunoaffinity chromatography combined with species-specific adsorption offering the highest specificity for complex applications.

What are the considerations for using these antibodies in detecting post-translational modifications?

When using Rabbit anti-Guinea Pig IgG secondary antibodies to detect primary antibodies targeting post-translational modifications (PTMs), several critical factors must be considered:

Epitope Accessibility Challenges:

• Fixation Impact on PTM Detection:
  Different fixation methods can preserve or mask PTMs:

  Fixation Method	Impact on PTM Preservation	Recommended for
  Paraformaldehyde (4%)	Preserves most PTMs but may reduce accessibility	Phosphorylation, acetylation
  Methanol	May disrupt some PTM epitopes	Methylation marks
  Glyoxal	Better preservation of nuclear proteins and their PTMs	Histone modifications

• Antigen Retrieval Requirements:
  PTM-specific epitopes often require optimized retrieval methods when using these secondary antibodies in IHC/IF applications.

Protocol Adaptations for PTM Detection:

• Signal Amplification Strategies:
  When detecting low-abundance PTMs:

  • Use HRP-conjugated Rabbit anti-Guinea Pig IgG with tyramide signal amplification

  • Apply fluorescent-conjugated antibodies with brighter fluorophores (e.g., DyLight 488)

  • Consider multiple-layer approaches for rare PTMs

• Cross-Linking Considerations:

  • Excessive fixation can mask PTMs or create artificial cross-links

  • Under-fixation may not preserve transient modifications

  • Titrate fixation conditions for each specific PTM

Validated Applications in PTM Research:

The utility of these antibodies in PTM detection has been demonstrated in recent research applications:

• Epigenetic Modifications:
  In CUT&Tag experiments, when paired with rabbit anti-H3K27me3 antibodies, guinea pig anti-rabbit IgG secondary antibodies successfully enabled profiling of this key histone modification .

• Phosphorylation Detection:
  When primary guinea pig antibodies target phosphorylated proteins, rabbit anti-guinea pig IgG antibodies provide sensitive detection with minimal background.

• Quality Control Metrics:
  For PTM detection, signal-to-noise ratio is particularly critical:

  • Strong signal in positive control samples (containing the PTM)

  • Minimal background in negative controls (e.g., dephosphorylated samples)

  • Clear distinction between modified and unmodified forms

These considerations ensure optimal detection of post-translational modifications when using Rabbit anti-Guinea Pig IgG antibodies as part of the detection system.

How do buffer compositions affect antibody performance in different applications?

Buffer composition significantly impacts the functionality, stability, and specificity of Rabbit anti-Guinea Pig IgG antibodies across different applications:

Standard Buffer Compositions and Their Effects:

Application-Specific Buffer Optimizations:

• Western Blotting:

  • Addition of 0.05-0.1% Tween-20 to wash and antibody dilution buffers reduces background

  • For milk-sensitive applications, switch to BSA-based blocking and dilution buffers

• Immunofluorescence:

  • Phosphate buffers must be free of autofluorescent contaminants

  • Addition of 0.05% Triton X-100 improves antibody penetration for intracellular targets

• ELISA:

  • Carbonate coating buffers (pH 9.6) for target adsorption

  • Tris-based (pH 7.4) antibody dilution buffers with 0.05% Tween-20

• CUT&RUN/CUT&Tag:

  • Specialized buffers containing spermidine (0.5 mM) and protease inhibitors

  • Digitonin permeabilization buffer for nuclear penetration

  • Ca²⁺-containing buffers (2 mM CaCl₂) for MNase activation

Reconstitution and Storage Buffer Impact:

Proper reconstitution is critical for maintaining antibody activity:

• "Reconstitute with deionized water (or equivalent)"

• "Let stand 30 minutes at room temperature to dissolve completely"

• "Centrifuge to remove any particulates"

For long-term storage preservation:

• "For extended storage aliquot contents and freeze at -20°C or below"

• "Avoid cycles of freezing and thawing"

• "Dilute with 50% glycerol and store at -20°C as a liquid"

These buffer considerations directly affect antibody performance metrics including specificity, sensitivity, and background levels across different experimental platforms.

How are Rabbit anti-Guinea Pig IgG antibodies advancing genomic research technologies?

Rabbit anti-Guinea Pig IgG antibodies have become instrumental in the development and optimization of cutting-edge genomic research technologies, particularly in chromatin profiling methods:

Contributions to Advanced Genomic Techniques:

• CUT&RUN (Cleavage Under Targets and Release Using Nuclease):
  These antibodies serve as crucial reagents for enhancing protein A binding capacity in CUT&RUN protocols. A recent validation by the Cantù Lab demonstrated that Guinea Pig anti-Rabbit IgG antibodies effectively amplify protein A binding sites when used with rabbit primary antibodies targeting histone modifications . The validated protocol successfully identified protein binding sites in human cell lines with significantly lower background compared to traditional ChIP-seq approaches.

• CUT&Tag (Cleavage Under Targets and Tagmentation):
  In CUT&Tag applications, these antibodies help bridge primary antibodies to the pA-Tn5 transposase fusion protein. Research from Tom Taghon's lab validated a protocol using rabbit anti-H3K27me3 primary antibodies with guinea pig anti-rabbit secondary antibodies for profiling H3K27me3 signals in human primary thymocytes and cell lines . This approach generated high-quality libraries with distinct nucleosomal patterns characteristic of H3K27me3 distribution.

• Multiplexed Epigenomic Profiling:
  The specificity of these antibodies enables simultaneous profiling of multiple chromatin features. Recent applications include multiplexed histone modification mapping and transcription factor binding site identification, facilitating comprehensive epigenomic characterization with reduced sample requirements.

Impact on Research Outcomes:

The implementation of these antibodies in genomic technologies has enabled several scientific advances:

• Recent publication: "Context-defined cancer co-dependency mapping identifies a functional interplay between PRC2 and MLL-MEN1 complex in lymphoma" in Nature Communications (2023) , which utilized CUT&RUN methodology with guinea pig anti-rabbit IgG antibodies.

• Another significant study: "H3K36 methylation maintains cell identity by regulating opposing lineage programmes" in Nature Cell Biology (2023) , which employed these antibodies for high-resolution chromatin state mapping.

These applications demonstrate how Rabbit anti-Guinea Pig IgG antibodies are advancing genomic research by enabling more sensitive, specific, and efficient chromatin profiling technologies compared to traditional approaches.

What emerging applications are being developed for these antibodies beyond traditional immunodetection?

Rabbit anti-Guinea Pig IgG antibodies are increasingly being applied in innovative research contexts beyond conventional immunodetection methods:

Emerging Applications:

• Super-Resolution Microscopy Techniques:
  The conjugation of these antibodies with photoactivatable fluorophores enables their use in techniques like:

  • STORM (Stochastic Optical Reconstruction Microscopy)

  • PALM (Photoactivated Localization Microscopy)

  • STED (Stimulated Emission Depletion)

  These approaches require highly specific secondary antibodies to achieve nanometer-scale resolution, with conjugates like DyLight 488 proving particularly effective .

• Proximity Ligation Assays (PLA):
  These antibodies can be adapted for PLA, where two proteins in close proximity can be detected through antibody-linked DNA amplification, revealing protein-protein interactions in situ with far greater sensitivity than co-immunoprecipitation.

• Antibody-Drug Conjugates for Research Models:
  Though primarily a research tool rather than therapeutic, these antibodies can be conjugated to toxins or cytostatic compounds for targeted elimination of specific cell populations in research models.

• Mass Cytometry (CyTOF) Applications:
  Conjugation with rare earth metals enables these antibodies to be used in mass cytometry, allowing simultaneous detection of over 40 parameters in single cells without fluorescence spectrum limitations.

• Tissue Clearing and 3D Imaging:
  These antibodies are being optimized for use with CLARITY, iDISCO, and other tissue clearing techniques that enable 3D visualization of intact tissues, where antibody penetration and specificity are critical factors.

• Microfluidic-Based Single-Cell Proteomics:
  Integration into microfluidic platforms for single-cell analysis, where the high specificity of these antibodies enables reliable protein quantification at the single-cell level.

Technical Adaptations Required:

For these emerging applications, specific technical modifications include:

• Conjugation with alternative labels (metal isotopes, DNA barcodes, etc.)

• Optimization of binding kinetics for microfluidic environments

• Enhanced stability for extended imaging sessions in super-resolution microscopy

• Minimized size (using F(ab) or F(ab')2 fragments) for better tissue penetration