Rabbit anti-Guinea Pig IgG Antibody;HRP conjugated

Product Specs

Buffer

0.01M Phosphate Buffered Saline (PBS), pH 7.4

Form

Liquid

Lead Time

Typically, we can ship products within 1-3 business days following receipt of your order. Delivery times may vary depending on the shipping method and destination. For specific delivery timelines, please contact your local distributor.

Q&A

What is the molecular basis for Rabbit anti-Guinea Pig IgG (H&L) HRP conjugated antibody specificity?

This secondary antibody's specificity derives from immunization of rabbits with purified guinea pig IgG whole molecules. The resulting polyclonal antibodies recognize epitopes on both heavy (H) and light (L) chains of guinea pig IgG. Immunoelectrophoresis analysis confirms this specificity, showing reactivity with heavy chains on guinea pig IgG and light chains on all guinea pig immunoglobulins, while showing no reactivity to non-immunoglobulin guinea pig serum proteins . The antibodies are typically purified through immunoaffinity chromatography using guinea pig IgG coupled to agarose beads, resulting in preparations with >95% purity as determined by SDS-PAGE .

How does the immunization and purification process affect antibody performance?

The production process significantly impacts antibody quality and performance characteristics:

Process Step	Details	Impact on Performance
Immunization	Rabbits hyperimmunized with guinea pig IgG	Generates high-titer, high-affinity antibodies
Affinity Purification	Using immobilized guinea pig IgG	Removes non-specific antibodies, reducing background
Conjugation	HRP enzyme attachment to purified IgG	Enables colorimetric/chemiluminescent detection

Antibodies that undergo more stringent purification steps generally show reduced background and higher signal-to-noise ratios in immunoassays . Researchers should consider the extent of purification and cross-adsorption when selecting antibodies for potentially cross-reactive experimental systems.

What are the optimal dilution ranges for different applications of Rabbit anti-Guinea Pig IgG-HRP conjugates?

Application	Recommended Dilution Range	Notes
Western Blot (ECL)	1:5,000 - 1:20,000	0.1-0.2 μg/ml final concentration
Western Blot (DAB)	1:1,500 - 1:5,000	0.7-3.3 μg/ml final concentration
ELISA	1:5,000 - 1:100,000	0.05-0.5 μg/ml final concentration
IHC-P/IHC-F	1:500 - 1:2,000	Higher concentrations typically needed for tissue sections
ICC/IF	1:500 - 1:2,000	Dilution may need optimization based on fixation method

Always prepare fresh working dilutions daily and validate for your specific experimental conditions. Titration experiments are recommended when using this antibody in a new application or with new sample types .

How should Rabbit anti-Guinea Pig IgG-HRP be stored to maintain optimal activity, and what reconstitution protocols are recommended?

Proper storage and reconstitution are critical for maintaining enzymatic activity and antibody functionality:

For lyophilized format:

Store unopened at 2-8°C. Long-term storage (years) is possible in this state .
For reconstitution, add 1.1 ml of sterile/deionized water to 1 mg of antibody (typically overfilled to ensure complete recovery) .
Allow to stand for 30 minutes at room temperature to dissolve completely.
Centrifuge to remove any particulates.

For long-term storage after reconstitution:

Dilute with glycerol to a final concentration of 50% glycerol (e.g., for 1.1 ml reconstituted antibody, add 1.1 ml of glycerol) .
Store as liquid at -20°C to prevent loss of enzymatic activity. This solution will not freeze at -20°C.
If using a 1:5000 dilution prior to adding glycerol, adjust to 1:2500 dilution after glycerol addition .

Important considerations:

Prepare working dilutions immediately before use and discard afterward .
Avoid repeated freeze/thaw cycles by preparing single-use aliquots .
Mix gently without foaming to avoid denaturing the antibody or creating bubbles that can interfere with pipetting .

What strategies can address high background or non-specific binding when using Rabbit anti-Guinea Pig IgG-HRP?

High background is a common challenge with HRP-conjugated secondary antibodies. Systematic troubleshooting approaches include:

Issue	Potential Causes	Optimization Strategies
Diffuse background	Insufficient blocking	Increase blocking time (1-2 hours); optimize blocker type (BSA, milk, commercial blockers)
Cross-reactivity	Antibody binding to non-target proteins	Use cross-adsorbed antibodies; increase washing steps or stringency
Speckled background	Antibody aggregation	Filter antibody solution; centrifuge before use; optimize antibody dilution
Edge effects in ELISA	Uneven temperature distribution	Equilibrate plates to room temperature; use plate sealers during incubations
Signal in negative controls	Endogenous peroxidase activity	Include peroxidase quenching step (3% H₂O₂ for 10 min) before antibody incubation

Additionally, incorporating a 0.05-0.1% Tween-20 in wash buffers can significantly reduce non-specific interactions while maintaining specific binding. For tissues with high endogenous biotin, which may cause background with avidin-biotin detection systems, consider using polymer-HRP detection systems instead .

How do buffer compositions affect the performance of Rabbit anti-Guinea Pig IgG-HRP in immunoassays?

Buffer composition significantly impacts antibody performance across different applications:

Buffer Component	Typical Concentration	Effect on Performance
Phosphate/Tris	10-50 mM	Maintains pH; optimal range 7.2-7.6 for most applications
NaCl	150 mM	Maintains ionic strength; reduces non-specific electrostatic interactions
BSA	0.1-1%	Blocks non-specific binding sites; stabilizes antibody
Tween-20	0.05-0.1%	Reduces hydrophobic interactions; may disrupt weak specific interactions if >0.1%
Proclin/Kathon	0.01-0.1%	Preservative; alternative to sodium azide (which inhibits HRP)
Glycerol	Up to 50%	Prevents freezing; stabilizes protein structure during storage

Important considerations:

Sodium azide should NEVER be used with HRP-conjugated antibodies as it irreversibly inhibits HRP activity, even at low concentrations .
For western blots, adding 0.1-0.5% non-fat dry milk to the antibody dilution buffer can further reduce background without compromising specific signal .
pH shifts outside the 7.0-7.6 range can dramatically reduce HRP activity and antibody binding efficiency .

How does epitope accessibility affect detection sensitivity when using Rabbit anti-Guinea Pig IgG-HRP in different immunoassay formats?

Epitope accessibility varies significantly across immunoassay formats and affects detection limits:

Assay Format	Epitope Accessibility	Optimization Strategies
Western Blot	Denatured proteins expose linear epitopes	Optimize SDS% and reducing agent concentration; consider non-reducing conditions for conformation-dependent antibodies
ELISA	Native conformation, potential steric hindrance	Use spacer arms in plate-coating protocols; optimize coating buffer pH
IHC-Paraffin	Epitope masking due to fixation	Implement heat-induced or enzymatic antigen retrieval; optimize retrieval buffer pH and time
IHC-Frozen	Better epitope preservation but potential structural damage	Optimize fixation method; use gentler permeabilization protocols
IF/ICC	Variable accessibility depending on fixation	Compare cross-linkers (PFA) vs. precipitating fixatives (methanol); adjust permeabilization

Research has demonstrated that detection sensitivity can vary by 2-10 fold between assay formats using the same antibody pair. For fixed tissues, epitope retrieval methods should be optimized specifically for the primary guinea pig antibody's target, as over-retrieval can destroy some epitopes while under-retrieval leaves others masked .

What are the mechanistic considerations for amplification systems to enhance sensitivity when using Rabbit anti-Guinea Pig IgG-HRP?

Several amplification systems can enhance the sensitivity of HRP-conjugated secondary antibodies:

Amplification System	Mechanism	Sensitivity Enhancement	Limitations
Tyramide Signal Amplification (TSA)	HRP catalyzes deposition of tyramide-fluorophores	10-50 fold	Potential background increase; requires optimization
Polymer-HRP Systems	Multiple HRP molecules per antibody	5-10 fold	May increase steric hindrance; reduced tissue penetration
Avidin-Biotin Complex (ABC)	Lattice formation of avidin-biotin-HRP	3-8 fold	Endogenous biotin interference; additional incubation steps
Enhanced Chemiluminescence (ECL)	Luminol oxidation with enhancers	10-100 fold for WB	Temporary signal; requires immediate imaging

For maximum sensitivity in low-abundance target detection, researchers should consider combining optimized antibody concentrations with appropriate amplification systems. For example, when detecting post-translational modifications of low-abundance proteins, a polymer-HRP system combined with enhanced chemiluminescence can provide detection limits in the picogram range, compared to standard HRP-DAB detection with sensitivity in the nanogram range .

How do different substrates for HRP affect sensitivity and dynamic range when using Rabbit anti-Guinea Pig IgG-HRP conjugates?

The choice of substrate significantly impacts assay performance characteristics:

Substrate	Detection Method	Sensitivity	Dynamic Range	Stability
DAB (3,3'-diaminobenzidine)	Colorimetric (brown)	0.5-5 ng	1-2 logs	Permanent, months to years
TMB (3,3',5,5'-tetramethylbenzidine)	Colorimetric (blue→yellow)	10-50 pg	2-3 logs	Hours (stop solution)
ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid)	Colorimetric (green)	0.1-1 ng	2 logs	Days (refrigerated)
ECL (enhanced chemiluminescence)	Luminescence	1-10 pg	3-4 logs	Minutes to hours
Fluorescent (Amplex Red)	Fluorescence	1-5 pg	3 logs	Hours (protected from light)

For quantitative applications like ELISA, TMB offers a good balance of sensitivity and working range. For western blots requiring maximum sensitivity, ECL substrates are preferred. For IHC applications where permanent staining is desired, DAB remains the standard despite lower sensitivity .

Researchers should note that substrate incubation time must be empirically determined for optimal signal-to-noise ratio, as extending incubation beyond optimal points typically increases background without proportionally increasing specific signal .

What are the critical control experiments required for validating results obtained with Rabbit anti-Guinea Pig IgG-HRP in different immunoassay formats?

Proper controls are essential for result validation and troubleshooting:

Control Type	Purpose	Implementation
Primary Antibody Omission	Controls for non-specific binding of secondary antibody	Process sample identically but omit primary guinea pig antibody
Isotype Control	Controls for non-specific binding due to Fc receptors	Use irrelevant guinea pig antibody of same isotype as primary
Absorption Control	Confirms primary antibody specificity	Pre-incubate primary antibody with purified antigen before staining
Tissue/Sample Negative Control	Controls for endogenous peroxidase/targets	Use known negative tissue or knockout/knockdown samples
Positive Control	Validates staining protocol	Use sample with known expression of target protein
Enzyme Activity Control	Ensures HRP activity	Include manufacturer's positive control or known positive sample

For western blots, loading controls (e.g., housekeeping proteins) should be included to normalize for protein loading variations. For ELISA, standard curves with known concentrations of analyte must be run in parallel with samples to enable quantification .

In multiplex assays where guinea pig antibodies are used alongside antibodies from other species, appropriate controls should verify the specificity of the rabbit anti-guinea pig IgG-HRP for guinea pig antibodies without cross-reactivity to other species' antibodies .

How can Rabbit anti-Guinea Pig IgG-HRP be optimized for multiplexed detection systems?

Optimizing for multiplexed detection requires careful consideration of several factors:

Factor	Optimization Strategy	Research Finding
Cross-reactivity	Use highly cross-adsorbed antibodies	Reduces background by >90% in multiplexed systems
Signal separation	Select compatible detection substrates	Precipitating vs. soluble substrates; different wavelength fluorescent products
Steric hindrance	Sequential rather than simultaneous detection	Can improve detection of closely located epitopes by 30-50%
Antibody concentration	Titrate each antibody individually	Optimal concentration may differ in multiplex vs. single-plex settings
Order of application	Apply lower-abundance target detection first	Can improve detection of minor components by 2-3 fold

Research has shown that for chromogenic multiplex IHC, using HRP-conjugated rabbit anti-guinea pig IgG with precipitating substrates like DAB (brown), followed by alkaline phosphatase systems with substrates like Fast Red (red) allows clear visual distinction of targets. For fluorescent multiplexing, using tyramide signal amplification systems with different fluorophores enables simultaneous detection of multiple targets with minimal cross-talk .

What are the methodological considerations when using Rabbit anti-Guinea Pig IgG-HRP in complex matrices or challenging samples?

Complex biological matrices and challenging samples require specialized approaches:

Sample Type	Challenge	Methodological Solution
Tissues with high endogenous peroxidase	False positive background	Quench with 0.3-3% H₂O₂ in methanol for 10-30 minutes before antibody incubation
Highly autofluorescent tissues	Signal interference	Use chromogenic rather than fluorescent detection; Sudan Black B treatment reduces autofluorescence
Tissues with high biotin content	Background with ABC systems	Use polymer-HRP detection or avidin/biotin blocking kits
Poorly fixed tissues	Inconsistent staining	Optimize fixation protocols; consider antigen retrieval methods
Low abundance targets	Weak signal	Implement signal amplification systems; increase primary antibody incubation time (overnight at 4°C)

Research findings indicate that for formalin-fixed paraffin-embedded (FFPE) tissues older than 5 years, extended antigen retrieval times (20-30 minutes vs. standard 10 minutes) combined with longer primary and secondary antibody incubations can improve detection sensitivity by 2-3 fold. For highly autofluorescent tissues like brain and kidney, copper sulfate treatment (10mM, 30 minutes) after secondary antibody incubation can reduce autofluorescence while preserving specific HRP signal .

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