Rabbit anti-Goat IgG Fc Antibody;FITC conjugated

Basic Research Questions

• What is the principle mechanism of Rabbit anti-Goat IgG Fc-FITC in immunological detection systems?

  Rabbit anti-Goat IgG Fc-FITC functions as a secondary detection reagent that specifically recognizes and binds to the Fc region of goat immunoglobulins. The antibody is generated by hyperimmunizing rabbits with purified goat IgG Fc fragments, followed by affinity purification to ensure specificity. The conjugated FITC (fluorescein isothiocyanate) molecule has an absorption maximum at 492-495 nm and emission maximum at 518-520 nm, producing a green fluorescence when excited with appropriate wavelength light .

  The binding occurs through specific molecular interactions between the antigen-binding region of the rabbit antibody and epitopes on the goat IgG Fc region. By targeting only the Fc region, these antibodies avoid interference with antigen-binding sites on primary goat antibodies, maintaining detection sensitivity. The fluorophore-to-protein ratio typically ranges from 3-7 moles FITC per mole antibody, optimized to provide sufficient signal without causing antibody inactivation through over-labeling .

• How should researchers determine the optimal dilution for Rabbit anti-Goat IgG Fc-FITC in different applications?

  Optimal dilution depends on multiple experimental factors including application type, target abundance, and detection system sensitivity. A methodical titration approach is recommended:

  Application	Recommended Starting Dilution Range	Optimization Parameters
  ELISA/FLISA	1:100 - 1:5000	Signal-to-noise ratio, background levels
  Flow Cytometry	1:50 - 1:500	Population separation, autofluorescence
  Immunohistochemistry	1:20 - 1:2000	Tissue type, fixation method
  Immunofluorescence	1:50 - 1:500	Cell type, fixation protocol
  Western Blot	1:100 - 1:500	Protein abundance, transfer efficiency

  Perform a series of 2-fold dilutions across this range and evaluate signal intensity versus background. For flow cytometry, include a fluorescence-minus-one (FMO) control. For microscopy applications, include a secondary-only control to assess non-specific binding. The optimal dilution will provide maximal specific signal with minimal background .

• What controls are essential when using Rabbit anti-Goat IgG Fc-FITC in experimental protocols?

  Robust experimental design requires incorporating multiple controls:

  • Isotype control: Use rabbit IgG-FITC (matched to the same isotype as the secondary antibody) to assess non-specific binding through Fc receptors .

  • Secondary-only control: Omit primary antibody but include Rabbit anti-Goat IgG Fc-FITC to detect non-specific binding of the secondary antibody to the sample .

  • Absorption control: Pre-incubate the secondary antibody with excess purified goat IgG to confirm binding specificity .

  • Biological negative control: Include samples known to be negative for the target antigen.

  • Fluorescence control: For multi-color experiments, single-stained controls are crucial to establish compensation parameters and identify spectral overlap .

  These controls allow discrimination between true positive signals and artifacts, especially critical in complex tissue samples or when examining low-abundance targets .

• How does storage and handling affect the performance of Rabbit anti-Goat IgG Fc-FITC antibodies?

  Proper storage significantly impacts antibody performance and shelf-life:

  • Temperature: Store at 2-8°C for short-term (≤1 month) or aliquot and freeze at -20°C with 50% glycerol for long-term storage .

  • Light exposure: FITC is particularly susceptible to photobleaching. Always protect from light using amber tubes or aluminum foil wrapping .

  • Freeze-thaw cycles: Minimize freeze-thaw cycles as they promote antibody aggregation and loss of activity. Prepare single-use aliquots before freezing .

  • Buffer composition: Phosphate buffered saline with 0.1% sodium azide helps maintain antibody stability. Avoid bacterial contamination .

  • Working dilutions: Prepare fresh working dilutions on the day of use for optimal performance .

  Performance can be monitored through regular quality control testing using standardized positive samples. A decline in fluorescence intensity or increased background may indicate antibody deterioration .

• What are the main differences between whole IgG, F(ab')2, and Fab fragment formats of Rabbit anti-Goat IgG Fc-FITC?

  Different antibody formats offer distinct advantages for specific applications:

  Format	Structure	Molecular Weight	Advantages	Best Applications
  Whole IgG	Complete antibody with Fc region	~160 kDa	Cost-effective, highest avidity	Standard applications, most immunoassays
  F(ab')2	Two Fab regions connected by disulfide bonds	~110 kDa	Eliminates Fc-mediated binding, reduces background	Tissues with Fc receptors, flow cytometry
  Fab	Single antigen-binding fragment	~50 kDa	Smallest size, best tissue penetration	Dense tissues, intracellular targets

  F(ab')2 fragments are generated by pepsin digestion under controlled conditions, cleaving the antibody below the hinge region to remove the Fc portion . This eliminates non-specific binding to Fc receptors present on many cell types, particularly important when working with immune cells or tissues with high Fc receptor expression . Whole IgG provides the strongest binding due to its bivalent nature, while Fab fragments offer superior tissue penetration but reduced avidity .