INSRR Antibody, FITC conjugated

What is INSRR and why is it important in metabolic research?

INSRR (Insulin Receptor-Related Receptor) is a member of the insulin receptor family that plays a crucial role in insulin signaling and glucose metabolism. It is a membrane-bound, single-pass type I membrane protein with a calculated molecular weight of approximately 144kDa . INSRR is essential for mediating insulin effects in various tissues and organs, contributing to metabolic homeostasis. Its dysregulation has been implicated in insulin resistance, type 2 diabetes, and other metabolic disorders . Understanding INSRR's expression patterns and signaling mechanisms provides critical insights into metabolic disease pathophysiology and potential therapeutic interventions.

What are the key applications for INSRR Antibody, FITC conjugated?

INSRR Antibody, FITC conjugated is primarily used in the following applications:

• Immunofluorescence (IF): For cellular localization studies in tissue sections or cultured cells

• Flow Cytometry (FC): For quantitative analysis of INSRR expression in cell populations

• Immunohistochemistry (IHC): For tissue distribution analysis in both frozen and paraffin-embedded specimens

• Western Blot (WB): Though less common with FITC-conjugated antibodies, specialized imaging systems can detect FITC signals on membranes

The specific applications may vary depending on the antibody's characteristics, with different antibodies showing variable reactivity across species including human, mouse, and rat samples .

How should FITC-conjugated antibodies be stored to maintain activity?

Proper storage is critical for maintaining FITC conjugate stability and activity:

• Store at -20°C in light-protected vials or covered with aluminum foil

• Avoid repeated freezing and thawing which can compromise both enzyme activity and antibody binding

• Conjugated antibodies are typically stable for at least 12 months at 4°C

• For longer storage (up to 24 months), dilute with up to 50% glycerol and store at -20°C to -80°C

• Always protect from light during storage and handling as FITC is photosensitive

Most commercial INSRR antibodies come in formulations containing stabilizers such as BSA (5 mg/mL) and preservatives like sodium azide (0.02%) in buffer solutions (commonly PBS, pH 7.4-7.6) .

How do I determine the optimal dilution for INSRR Antibody, FITC conjugated in my experiment?

Determining optimal dilution requires methodical titration:

• Begin with the manufacturer's suggested dilution range (typically 1:20-1:100 for fluorescently-labeled antibodies)

• Prepare a series of dilutions (e.g., 1:20, 1:50, 1:100, 1:200) for your specific sample type

• Run parallel experiments with positive controls (tissues known to express INSRR, such as rat kidney, stomach, or pancreas)

• Include negative controls:

  • Secondary antibody only (no primary)

  • Isotype control antibody

  • Samples known not to express INSRR

• Evaluate signal-to-noise ratio at each dilution

• Select the dilution that provides optimal specific signal with minimal background

• Validate with additional independent experiments

For Western blot applications, recommended dilutions typically range from 1:500 to 1:1000, but this should be optimized for FITC-conjugated variants .

What controls should be included when using INSRR Antibody, FITC conjugated?

A robust experimental design requires multiple controls:

The choice of control tissues is crucial, as INSRR expression varies significantly between tissues and species. Reactivity has been documented across human, mouse, rat, dog, guinea pig, horse, and zebrafish samples with varying homology .

How can I design multi-parameter experiments using INSRR Antibody, FITC conjugated with other fluorophores?

For complex co-localization studies, consider these methodological approaches:

• Spectral Compatibility: FITC (excitation max: 492nm, emission max: 520nm) pairs well with fluorophores having minimal spectral overlap such as:

  • DAPI (nuclear stain)

  • Cy3/TRITC (for second protein of interest)

  • APC (for third protein of interest)

• Sequential Staining Protocol:

  • Begin with the weakest signal antibody (often FITC-conjugated)

  • Use careful washing steps between applications (3×5 min with PBS + 0.05% Tween-20)

  • Apply stronger fluorophore-conjugated antibodies subsequently

  • Include compensation controls for flow cytometry applications

• Cross-Reactivity Prevention:

  • Use antibodies raised in different host species

  • Consider using F(ab) fragments to prevent Fc receptor binding

  • Block with 5% normal serum from the host species of secondary antibodies

• Signal Amplification Options:

  • Use biotinylated anti-FITC antibody followed by streptavidin-FITC for increased sensitivity

  • Apply tyramide signal amplification systems compatible with FITC

When designing these experiments, carefully document the spectral properties of all fluorophores and verify no cross-reactivity between secondary antibodies.

What approaches can minimize photobleaching of FITC-conjugated antibodies in long-duration imaging?

FITC is particularly susceptible to photobleaching. Implement these methodological solutions:

• Anti-fade Reagents:

  • Use commercial anti-fade mounting media specifically formulated for FITC

  • Consider oxygen-scavenging systems (e.g., glucose oxidase/catalase)

• Imaging Parameters Optimization:

  • Reduce exposure time and intensity to minimum required for detection

  • Increase camera gain/sensitivity rather than excitation intensity

  • Use neutral density filters to attenuate excitation light

  • Employ confocal apertures to minimize out-of-focus excitation

• Advanced Microscopy Approaches:

  • Consider time-lapse capture with defined intervals rather than continuous exposure

  • Use deconvolution to extract maximum information from lower-intensity images

  • Implement resonant scanning for faster image acquisition with reduced light exposure

• Sample Preparation Enhancements:

  • Add 1-4 diazabicyclo octane (DABCO) to mounting medium

  • Store slides at 4°C in the dark when not imaging

  • Seal coverslips completely to prevent oxidation

For quantitative studies, capture reference samples at standardized timepoints to normalize for any photobleaching effects.

How can I address high background when using INSRR Antibody, FITC conjugated?

High background is a common challenge with FITC-conjugated antibodies. Implement this systematic approach:

• Optimize Blocking:

  • Increase blocking concentration (try 5-10% BSA or normal serum)

  • Extend blocking time to 2 hours at room temperature

  • Add 0.1-0.3% Triton X-100 for membrane permeabilization when appropriate

• Antibody Dilution Adjustments:

  • Further dilute primary antibody (try 2-5× higher dilution)

  • Prepare antibody dilutions in blocking buffer containing 1% BSA

  • Pre-absorb antibody with tissue powder from non-expressing samples

• Washing Modifications:

  • Increase wash duration (5 × 10 minutes)

  • Use 0.05-0.1% Tween-20 in wash buffer

  • Consider adding 150-500 mM NaCl to wash buffer to reduce non-specific ionic interactions

• Tissue-Specific Treatments:

  • For tissues with high autofluorescence, pretreat with 0.1-1% sodium borohydride

  • Include 10-50 mM NH₄Cl in wash buffer to reduce aldehyde-induced background

  • For tissue sections, incorporate a Sudan Black B treatment (0.1-0.3% in 70% ethanol)

The suggested dilution range of 1:20-1:100 provides a starting point, but extensive optimization may be required for specific tissue types .

What are potential causes for weak or no signal when using INSRR Antibody, FITC conjugated?

When INSRR detection yields weak or absent signals, investigate these methodological factors:

• Antibody Activity Assessment:

  • Verify fluorophore integrity (FITC has excitation/emission maxima at 492/520nm)

  • Test antibody with a known positive control (e.g., rat kidney)

  • Check antibody storage conditions (light exposure, freeze-thaw cycles)

  • Confirm antibody concentration (typically 0.5-1.0 mg/ml for commercial preparations)

• Antigen Retrieval Optimization:

  • For formalin-fixed tissues, try heat-induced epitope retrieval (citrate buffer, pH 6.0)

  • Consider alternative retrieval methods (Tris-EDTA, pH 9.0; enzymatic digestion)

  • Extend retrieval time incrementally (10, 20, 30 minutes)

• Fixation Impact Analysis:

  • Different fixatives may mask the INSRR epitope

  • Consider testing paraformaldehyde, methanol, and acetone fixation

  • Reduce fixation time if overfixation is suspected

• Signal Amplification Strategies:

  • Implement two-layer detection using anti-FITC antibody followed by FITC-conjugated secondary

  • Use tyramide signal amplification compatible with fluorescence

  • Consider enzyme-mediated amplification systems

If the antibody recognizes a specific amino acid sequence (e.g., AA 747-921 of human INSRR), verify that this region is conserved in your experimental species and not masked by protein folding or post-translational modifications .

How can I accurately quantify INSRR expression levels using FITC-conjugated antibodies?

Quantitative analysis requires standardized methodology:

• Flow Cytometry Approach:

  • Use calibration beads with known FITC molecules per bead to create standard curves

  • Apply consistent voltage settings across experiments

  • Report results as Molecules of Equivalent Soluble Fluorochrome (MESF)

  • Include fluorescence-minus-one (FMO) controls for accurate gating

• Immunofluorescence Quantification:

  • Capture images with identical exposure settings

  • Analyze using specialized software (ImageJ/FIJI, CellProfiler)

  • Normalize FITC signal to cell number or tissue area

  • Implement thresholding based on negative control samples

• Western Blot Detection (specialized systems):

  • Include concentration curve of recombinant INSRR protein

  • Apply standard curve fitting for quantification

  • Normalize to loading controls (beta-actin, GAPDH)

• Statistical Analysis Considerations:

  • Apply appropriate transformations to achieve normal distribution

  • Use ANOVA with post-hoc tests for multiple comparison analysis

  • Include biological replicates (n≥3) to account for variation

The expected molecular weight of 144kDa for INSRR should be used as confirmation of specificity in Western blot applications .

How do the binding characteristics of different region-specific INSRR antibodies compare?

Different INSRR antibodies target distinct epitopes, affecting their performance:

When designing experiments targeting specific INSRR domains or phosphorylation states, select antibodies recognizing the appropriate region. Consider using multiple antibodies targeting different epitopes for confirmation of specificity, particularly in tissues with complex protein expression patterns.

For immunoprecipitation studies followed by mass spectrometry, antibodies targeting regions with minimal post-translational modifications should be selected to maximize pull-down efficiency.

Each antibody should be validated in the specific experimental system being studied, as epitope accessibility may vary depending on protein conformation, interactions, and modifications.