Rabbit anti-Human IgG Fc Antibody;FITC conjugated

How are Rabbit anti-Human IgG Fc FITC-conjugated antibodies prepared?

These antibodies are typically prepared through a multi-step process:

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

• Purification: The antibodies are purified from rabbit antiserum using immunoaffinity chromatography with human IgG coupled to agarose beads

• Additional purification: Solid phase adsorption(s) may be performed to remove unwanted reactivities

• Conjugation: The purified antibodies are conjugated with FITC (Fluorescein-5-isothiocyanate) under optimal conditions

This process yields antibodies with a typical fluorophore/protein ratio of approximately 7.5 μg FITC per mg of antibody, or 2.8 moles of FITC per mole of IgG .

What are the recommended applications for Rabbit anti-Human IgG Fc FITC antibodies?

These antibodies are validated for multiple applications including:

The versatility of these conjugates makes them suitable for numerous immunodetection protocols in research settings .

What are the proper storage conditions for FITC-conjugated antibodies?

To maintain optimal activity, Rabbit anti-Human IgG Fc FITC antibodies require specific storage conditions:

• Storage temperature: 4°C for short-term (weeks) or -20°C for long-term storage

• Protect from light: FITC is sensitive to photobleaching, requiring storage in dark conditions

• Avoid freeze-thaw cycles: Repeated freezing and thawing should be avoided

• Formulation: Often supplied as lyophilized powder requiring reconstitution or as liquid in buffer containing preservatives

• Extended shelf-life: Adding glycerol to a final concentration of 50% can extend shelf-life when stored at -20°C

After reconstitution or dilution, these antibodies should ideally be used within 24 hours for optimal performance .

How can I optimize signal-to-noise ratio when using FITC-conjugated antibodies in fluorescence microscopy?

Optimizing signal-to-noise ratio requires a systematic approach:

• Sample preparation:

  • Use freshly prepared 4% paraformaldehyde for fixation

  • Implement thorough blocking (5% BSA in PBS or 10% serum from the same species as the secondary antibody)

  • Include 0.1-0.3% Triton X-100 for permeabilization when detecting intracellular antigens

• Antibody optimization:

  • Perform titration experiments to determine optimal antibody concentration

  • Typical working dilutions range from 1:1,000 to 1:5,000 for fluorescence microscopy

  • Increase incubation time (up to overnight at 4°C) with more dilute antibody solutions

• Washing protocols:

  • Implement stringent washing (at least 3 × 5 minutes) with PBS-T (PBS + 0.05% Tween-20)

  • Include a high-salt wash step (PBS with 500mM NaCl) to reduce non-specific ionic interactions

• Mounting considerations:

  • Use anti-fade mounting media containing DAPI for nuclear counterstaining

  • Seal slides with nail polish to prevent drying and oxidation

• Microscopy settings:

  • Use appropriate excitation (492nm) and emission (520nm) filter sets

  • Implement background subtraction in image analysis

These strategies collectively enhance specific signal while minimizing background fluorescence .

What controls should be included when using Rabbit anti-Human IgG Fc FITC antibodies?

A comprehensive experimental design should include these essential controls:

• Isotype control:

  • FITC-conjugated rabbit IgG of the same isotype but lacking specificity for human IgG

  • Controls for non-specific binding of the antibody constant region

• Secondary antibody-only control:

  • Samples processed identically but without primary antibody

  • Identifies background from non-specific secondary antibody binding

• Blocking validation:

  • Pre-incubation of the antibody with purified human IgG

  • Should abolish specific staining while leaving non-specific background unchanged

• Positive control:

  • Tissues or cells known to express human IgG (e.g., human peripheral blood B cells)

  • Confirms antibody functionality under experimental conditions

• Negative control:

  • Tissues or cells known not to express human IgG

  • Identifies false positive signals

• Spectral controls:

  • Samples labeled with individual fluorophores when performing multiplex experiments

  • Essential for compensation in flow cytometry or correction of spectral overlap in microscopy

These controls enable confident interpretation of experimental results by distinguishing specific from non-specific signals .

What factors affect the binding efficiency of Rabbit anti-Human IgG Fc FITC antibodies in experimental settings?

Several critical factors influence binding efficiency:

• Epitope accessibility:

  • Fixation methods can mask epitopes (formaldehyde cross-linking may reduce accessibility)

  • Antigen retrieval may be necessary for formalin-fixed paraffin-embedded (FFPE) tissues

  • Sample permeabilization affects intracellular epitope access

• Sample-related factors:

  • pH of incubation buffer (optimal range typically 7.2-7.4)

  • Ionic strength of solutions affects antibody-antigen interactions

  • Presence of detergents may enhance or inhibit binding

  • Blocking reagents can interfere with specific binding if used at excessive concentrations

• Antibody-related factors:

  • F/P ratio (fluorophore/protein) affects binding and fluorescence intensity

  • Typical optimal ratio is 2.8 moles FITC per mole IgG

  • Over-conjugation can impair antibody function

  • Antibody concentration must be optimized for each application

• Experimental conditions:

  • Temperature (4°C for reduced non-specific binding, room temperature for faster kinetics)

  • Incubation time (longer for dilute antibodies or challenging samples)

  • Washing stringency affects signal-to-noise ratio

• Technical considerations:

  • FITC photobleaching during extended imaging sessions

  • Degradation of antibodies during storage

  • Freeze-thaw cycles can reduce activity

Understanding these factors enables researchers to optimize protocols for specific experimental requirements .

How can Rabbit anti-Human IgG Fc FITC antibodies be used in multiplex immunofluorescence experiments?

Multiplexing strategies with FITC-conjugated antibodies require careful planning:

• Spectral considerations:

  • FITC excitation maximum: 492nm

  • FITC emission maximum: 520nm

  • Compatible fluorophores include TRITC/Texas Red (red), Cy5 (far-red), and DAPI (blue)

  • Minimal spectral overlap when using appropriate filter sets

• Sequential staining approach:

  • Use primary antibodies from different host species

  • Apply species-specific secondary antibodies sequentially

  • Include blocking steps between sequences to prevent cross-reactivity

• Direct conjugate multiplexing:

  • Combine with other directly conjugated antibodies (e.g., PE, APC)

  • Requires careful titration of each conjugate

  • Particularly useful for flow cytometry applications

• Tyramide signal amplification (TSA):

  • Compatible with FITC-conjugated antibodies

  • Allows multiple antibodies from same species to be used sequentially

  • Requires HRP-conjugated secondary antibodies and FITC-tyramide

• Technical considerations:

  • Implement proper compensation controls in flow cytometry

  • Use linear unmixing for confocal microscopy with spectral detection

  • Consider photobleaching rates during sequential imaging

These approaches enable simultaneous detection of multiple targets, increasing the information obtained from a single specimen .

How can I validate and troubleshoot specificity issues with Rabbit anti-Human IgG Fc FITC antibodies?

A systematic approach to validation and troubleshooting includes:

• Specificity validation:

  • Western blot with purified human IgG should show bands at ~50kDa (heavy chain)

  • Immunoprecipitation followed by mass spectrometry can confirm target identity

  • Cross-adsorption testing against other immunoglobulin classes confirms Fc specificity

  • Immunoelectrophoresis should show a single precipitin arc against human IgG

• High background troubleshooting:

  • Increase blocking time and concentration (5% BSA or 10% serum)

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Include 0.1-0.5M NaCl in washing buffers to reduce ionic interactions

  • Centrifuge antibody solution before use to remove aggregates

• Weak signal troubleshooting:

  • Verify antibody activity with a dot blot of purified human IgG

  • Optimize fixation protocol (overfixation can mask epitopes)

  • Implement antigen retrieval for FFPE samples

  • Increase antibody concentration or incubation time

  • Check for photobleaching of FITC during long imaging sessions

• Cross-reactivity assessment:

  • Test against purified immunoglobulins from different species

  • Validate against tissues from IgG-knockout models if available

  • Pre-adsorb antibody with potential cross-reactive proteins

• Instrument validation:

  • Verify proper function of excitation (492nm) and emission (520nm) filters

  • Check laser/light source intensity

  • Use calibration beads to validate instrument performance

These validation steps ensure experimental reliability and facilitate troubleshooting when issues arise .

How do Rabbit anti-Human IgG Fc FITC antibodies perform in quantitative flow cytometry experiments?

For quantitative flow cytometry applications:

• Standardization requirements:

  • Use calibration beads with known quantities of fluorochrome

  • Convert fluorescence intensity to Molecules of Equivalent Soluble Fluorochrome (MESF)

  • Determine antibody binding capacity using standards with known quantities of target molecules

• Optimization for quantitative analysis:

  • Maintain consistent antibody lot and concentration between experiments

  • Standardize cell numbers (typically 5μl per million cells in 100μl volume)

  • Implement rigid fixation and permeabilization protocols

  • Use consistent instrument settings, validated with calibration beads

• Data analysis considerations:

  • Apply appropriate compensation for spectral overlap

  • Use median fluorescence intensity (MFI) rather than mean for non-normal distributions

  • Convert raw fluorescence to absolute numbers using calibration curves

  • Account for autofluorescence with unstained controls

• Limitations and considerations:

  • FITC brightness is pH-sensitive (optimal at pH ≥7.4)

  • Photobleaching during extended sample handling can affect quantitation

  • F/P ratio variations between lots affect signal intensity

  • Standard curves should be generated for each experimental session

• Advanced techniques:

  • Quantum dot calibration for enhanced quantitation precision

  • Comparison of surface IgG levels between different cell populations

  • Kinetic measurements of antibody binding

These approaches enable robust quantitative analysis of human IgG expression in research samples .

What is the specificity of Rabbit anti-Human IgG Fc Antibodies?

Rabbit anti-Human IgG Fc antibodies specifically recognize and bind to the constant (Fc) region of human immunoglobulin G. These antibodies react with the heavy chains of human IgG but not with the light chains on most human immunoglobulins . Immunoelectrophoresis testing typically shows a single precipitin arc against anti-Fluorescein, anti-Rabbit Serum, Human IgG, and Human Serum . Most preparations do not cross-react with human IgM, IgA, or non-immunoglobulin serum proteins, though some cross-reactivity with immunoglobulins from other species may occur . Clone-specific differences in reactivity exist - for example, clone M1310G05 demonstrates stronger affinity for IgG1 and IgG3 than for IgG2 and IgG4 .

What are the structural and fluorescent properties of FITC-conjugated antibodies?

FITC (Fluorescein-5-isothiocyanate) is a small fluorescent molecule that is covalently attached to the antibody protein through reaction with primary amines. These conjugates have specific spectral properties:

The fluorescence emission appears green when visualized by fluorescence microscopy with appropriate filter sets . The conjugation process is carefully controlled to maintain antibody functionality while providing sufficient fluorescence signal. Over-conjugation (too many FITC molecules per antibody) can adversely affect antibody binding capacity, while under-conjugation results in weak fluorescence signals .

What applications are Rabbit anti-Human IgG Fc FITC antibodies validated for?

These antibodies are validated for multiple immunological techniques:

The antibodies perform well in these applications because of their high specificity for human IgG and the bright fluorescence signal provided by the FITC conjugate . Each application requires specific optimization of antibody concentration, incubation conditions, and detection parameters.

What are the proper storage and handling recommendations for maintaining antibody activity?

To preserve the functional and fluorescent properties of Rabbit anti-Human IgG Fc FITC antibodies:

• Store at 4°C for short-term (weeks) or -20°C for long-term storage

• Protect from light exposure using amber tubes or aluminum foil wrapping

• Avoid repeated freeze-thaw cycles that can denature the antibody protein

• For lyophilized products, reconstitute with the recommended volume of distilled water (typically to 1.5 mg/mL)

• After reconstitution, centrifuge if the solution is not completely clear

• For extended shelf-life, add glycerol to 50% final concentration and store at -20°C

• Use reconstituted antibody within several weeks if stored at 4°C as an undiluted liquid

• After dilution for working solutions, use within 24 hours for optimal performance

These storage and handling practices are critical for maintaining both the immunological specificity and fluorescent properties of the conjugate .

How can I optimize signal-to-noise ratio when using FITC-conjugated antibodies in immunofluorescence applications?

Optimizing signal-to-noise ratio requires systematic protocol refinement:

• Sample preparation optimization:

  • Use gentle fixation methods (2-4% PFA for 10-15 minutes) to preserve epitope accessibility

  • Implement thorough permeabilization for intracellular targets (0.1-0.3% Triton X-100)

  • Block with 5% BSA in PBS or 10% serum from the same species as the secondary antibody

  • Include 0.1% Tween-20 in blocking buffers to reduce hydrophobic interactions

• Antibody titration:

  • Perform systematic dilution series to determine optimal concentration

  • Test 1:500, 1:1000, 1:2000, 1:5000 dilutions for microscopy applications

  • For flow cytometry, evaluate 5 μl per million cells in 100 μl staining volume

• Advanced washing strategies:

  • Increase number of washes (minimum 3×5 minutes)

  • Include detergent (0.05-0.1% Tween-20) in wash buffers

  • Consider high-salt wash steps (500mM NaCl) to disrupt non-specific ionic interactions

  • Use gentle agitation during washing to enhance removal of unbound antibody

• Mounting and imaging considerations:

  • Use anti-fade mounting media containing DAPI for nuclear counterstaining

  • Implement appropriate excitation (492nm) and emission (520nm) filter sets

  • Adjust exposure times to minimize photobleaching while maintaining adequate signal

  • Consider deconvolution algorithms to enhance signal-to-noise ratio post-acquisition

These strategies collectively minimize background while preserving specific fluorescence signals .

What are the molecular mechanisms of Rabbit anti-Human IgG Fc antibody binding and how do these affect experimental design?

Understanding the molecular interactions between these antibodies and their targets is crucial for experimental design:

• Structural basis of recognition:

  • The Fc region of human IgG contains conserved domains (CH2 and CH3)

  • Rabbit anti-Human IgG Fc antibodies recognize conformational epitopes within these domains

  • Recognition is largely independent of the variable regions or antigen binding sites

  • FITC conjugation typically occurs on lysine residues away from antigen-binding sites

• Impact on experimental design:

  • Sample preparation must preserve the native conformation of the Fc domain

  • Harsh fixation or denaturing conditions can disrupt epitope recognition

  • Polyclonal preparations recognize multiple epitopes, providing signal amplification

  • Monoclonal antibodies like clone M1310G05 target specific epitopes with subclass preferences

• Binding kinetics considerations:

  • Binding affinity affects optimal incubation times and temperatures

  • Higher-affinity antibodies may require shorter incubation periods

  • Temperature affects both reaction kinetics and potential non-specific interactions

  • Room temperature incubation accelerates binding but may increase background

• Cross-reactivity management:

  • Pre-adsorption of antibodies removes unwanted reactivities

  • Solid-phase adsorption with related immunoglobulins can enhance specificity

  • Using F(ab')2 fragments instead of whole IgG can reduce Fc-mediated interactions

Understanding these molecular interactions enables rational optimization of immunodetection protocols .

What controls are essential when using Rabbit anti-Human IgG Fc FITC antibodies in research applications?

Robust experimental design requires multiple controls:

• Isotype controls:

  • FITC-conjugated rabbit IgG lacking specificity for human IgG

  • Controls for non-specific binding through Fc receptors or hydrophobic interactions

  • Should be used at the same concentration as the specific antibody

• Blocking validation controls:

  • Pre-incubation of antibody with purified human IgG

  • Should abolish specific staining while leaving background unchanged

  • Competitive inhibition confirms binding specificity

• Cellular/tissue controls:

  • Positive controls: Human B lymphocytes or plasma cells (natural IgG expressors)

  • Negative controls: Non-human tissues or IgG-negative cell lines

  • Processing controls: Samples subjected to identical protocols except for primary antibody

• Technical controls:

  • Autofluorescence control: Unstained sample to establish baseline fluorescence

  • Single-stain controls: Essential for compensation in multicolor flow cytometry

  • Fluorescence-minus-one (FMO) controls: For accurate gating in flow cytometry

• Antibody validation controls:

  • Western blot or ELISA against purified human IgG to confirm specificity

  • Cross-adsorption testing against other immunoglobulin classes

  • Immunoelectrophoresis should show a single precipitin arc against human IgG

These controls collectively ensure experimental reliability and facilitate accurate interpretation of results .

How can Rabbit anti-Human IgG Fc FITC antibodies be integrated into multiplex immunodetection systems?

Multiplexing strategies require careful consideration of spectral properties and antibody compatibility:

• Spectral multiplexing approaches:

  • FITC (excitation: 492nm, emission: 520nm) can be combined with spectrally distinct fluorophores

  • Compatible fluorophores include TRITC/Texas Red (red), Cy5 (far-red), and DAPI (blue)

  • Modern flow cytometers can resolve FITC from PE, PE-Cy5, APC, and other fluorophores

  • Confocal microscopy with spectral detection enables separation of overlapping emissions

• Multi-epitope detection strategies:

  • Combine with primary antibodies from different host species

  • Use species-specific secondary antibodies with different fluorophores

  • Implement sequential staining with blocking between steps to prevent cross-reactivity

  • Tyramide signal amplification (TSA) allows multiple antibodies from same species

• Technical considerations for flow cytometry:

  • Follow standardized staining protocols (5 μl antibody per million cells)

  • Include single-stain controls for accurate compensation

  • Use compensation beads for consistent settings across experiments

  • Apply Boolean gating strategies for complex phenotyping

• Advanced imaging multiplexing:

  • Sequential imaging to minimize photobleaching of sensitive fluorophores

  • Linear unmixing algorithms for separating overlapping emission spectra

  • Consider antibody stripping and re-probing for highly multiplexed imaging

  • Automated image analysis for colocalization quantification

These approaches enable simultaneous detection of multiple targets, maximizing the information obtained from limited samples .

What are the emerging advanced applications of Rabbit anti-Human IgG Fc FITC antibodies in cutting-edge research?

These antibodies are finding utility in innovative research applications:

• Super-resolution microscopy:

  • FITC conjugates are compatible with structured illumination microscopy (SIM)

  • Can be used in stimulated emission depletion (STED) microscopy with appropriate imaging parameters

  • Direct stochastic optical reconstruction microscopy (dSTORM) applications require special buffers

  • Resolution improvements from ~250nm to ~20-100nm enable visualization of nanoscale structures

• Microfluidic and single-cell applications:

  • Integration with microfluidic antibody capture assays

  • Single-cell secretion profiling using antibody-coated surfaces

  • Droplet-based single-cell analysis systems for high-throughput screening

  • Correlation of surface IgG expression with transcriptomic profiles

• In vivo imaging applications:

  • Near-infrared derivatives for deeper tissue penetration

  • Conjugation to nanoparticles for multimodal imaging

  • Intravital microscopy of antibody-producing cells in living organisms

  • Correlative light and electron microscopy using FITC pre-embedding

• Quantitative applications:

  • Standardized beads for absolute quantification of surface IgG expression

  • Quantum dot calibration for enhanced precision

  • Automated image analysis algorithms for objective quantification

  • Machine learning approaches for complex pattern recognition

These emerging applications demonstrate the continued utility of these antibodies in pushing the boundaries of immunological research .

How can inconsistent results with Rabbit anti-Human IgG Fc FITC antibodies be systematically troubleshooted?

A structured troubleshooting approach should address multiple potential issues:

• Antibody quality assessment:

  • Verify antibody concentration and F/P ratio (ideally 2.8 moles FITC per mole IgG)

  • Check for precipitates or aggregation (centrifuge before use if necessary)

  • Confirm fluorescence using a simple dot blot with purified human IgG

  • Assess activity against a standardized positive control

• Protocol parameter optimization:

  • Fixation: Test multiple fixatives and durations to preserve epitope accessibility

  • Blocking: Evaluate different blocking agents (BSA, serum, commercial blockers)

  • Incubation conditions: Compare room temperature vs. 4°C, different durations

  • Washing stringency: Assess buffer composition, number and duration of washes

• Sample-related troubleshooting:

  • Autofluorescence mitigation: Test sodium borohydride or Sudan Black treatment

  • Fc receptor blocking: Include normal rabbit IgG to block Fc receptors

  • Endogenous biotin blocking: Use avidin/biotin blocking kits if required

  • Antigen retrieval: Evaluate heat-induced or enzymatic retrieval methods

• Instrument and detection troubleshooting:

  • Filter set verification: Ensure proper excitation (492nm) and emission (520nm) filters

  • Detector sensitivity: Adjust PMT voltage or exposure time settings

  • Signal amplification: Consider tyramide signal amplification for weak signals

  • Photobleaching: Minimize exposure time, use anti-fade reagents

• Antibody-specific issues:

  • Cross-reactivity: Test on negative control samples to assess specificity

  • Lot variation: Compare performance between lot numbers

  • Storage degradation: Verify proper storage conditions were maintained

  • Antibody concentration: Perform systematic titration experiments

This systematic approach ensures identification and resolution of technical issues affecting experimental reliability .