GRPR Antibody, FITC conjugated

What is GRPR and why is it an important research target?

Gastrin-releasing peptide receptor (GRPR, also known as GRP-R or GRP-preferring bombesin receptor) is a receptor that mediates the effects of gastrin-releasing peptide (GRP) in various physiological systems. GRP regulates numerous functions in the gastrointestinal and central nervous systems, including release of gastrointestinal hormones, smooth muscle cell contraction, and epithelial cell proliferation . GRPR is particularly important in neuroscience research as it plays a significant role in mediating nonhistaminergic itch transmission in the spinal cord . The receptor has also been identified as a potential target in certain neoplastic conditions, as GRP is a potent mitogen for neoplastic tissues .

What is the difference between GRPR antibodies and GRPR-FITC conjugated antibodies?

GRPR antibodies are immunoglobulins specifically designed to recognize and bind to GRPR proteins. Unconjugated GRPR antibodies require a secondary detection system when used in techniques like immunohistochemistry or flow cytometry. In contrast, GRPR antibodies conjugated with FITC have the fluorophore (Fluorescein Isothiocyanate) directly attached to the antibody molecule . This conjugation allows for direct visualization of the antibody binding without requiring a secondary antibody, simplifying protocols and reducing background in fluorescence-based applications. FITC emits green fluorescence when excited with the appropriate wavelength, making it useful for applications like immunofluorescence microscopy, flow cytometry, and fluorescence-based ELISAs .

What are the recommended storage conditions for GRPR antibody, FITC conjugated?

GRPR antibodies conjugated with FITC should be stored under specific conditions to maintain their functionality. Upon receipt, store the antibody at -20°C or -80°C to preserve integrity . It's important to avoid repeated freeze-thaw cycles as this can compromise antibody function. For conjugated antibodies specifically, light protection is critical to prevent photobleaching of the FITC fluorophore. They should be stored in light-protected vials or covered with a light-protecting material such as aluminum foil .

For long-term storage (up to 24 months), conjugated antibodies may be diluted with up to 50% glycerol and stored at -20°C to -80°C . It's worth noting that freezing and thawing conjugated antibodies can compromise both enzyme activity and antibody binding capability . Some products are formulated with 50% glycerol and other stabilizers in their buffer system, which helps maintain antibody integrity during storage .

What applications are GRPR-FITC antibodies validated for?

GRPR antibodies conjugated with FITC have been validated for several experimental applications, with varying utility depending on the specific product. Based on the search results, these applications include:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of GRPR in solution-based samples

• WB (Western Blotting): For detecting GRPR in protein lysates separated by electrophoresis

• IF (Immunofluorescence): For visualizing GRPR localization in cells and tissues using fluorescence microscopy

• IHC (Immunohistochemistry): For detecting GRPR in tissue sections

When designing experiments, it's important to verify that the specific antibody you're using has been validated for your intended application. Some antibodies may perform better in certain applications than others, depending on their epitope recognition, affinity, and specificity.

How should I design controls when using GRPR-FITC antibodies in immunofluorescence studies?

Proper controls are essential when using GRPR-FITC antibodies in immunofluorescence studies to ensure reliable and interpretable results:

• Negative Controls:

  • Isotype control: Use a non-specific FITC-conjugated antibody of the same isotype and host species (e.g., rabbit IgG-FITC)

  • Secondary antibody only control (if using an additional detection system)

  • Samples known to be negative for GRPR expression

  • Omission of primary antibody to assess background fluorescence

• Positive Controls:

  • Tissues or cell lines with known GRPR expression

  • Recombinant GRPR protein (similar to the immunogen used to generate the antibody)

• Blocking Controls:

  • Pre-incubation of the antibody with its specific blocking peptide (e.g., Catalog # AAP76226 for specific anti-GRPR antibodies)

  • This helps confirm specificity of staining

• Cross-reactivity Controls:

  • If studying multiple bombesin receptor family members (GRPR, NMBR), ensure your GRPR antibody doesn't cross-react with related proteins

Additionally, when analyzing co-localization with other markers, appropriate filter sets should be used to prevent bleed-through of FITC fluorescence into other channels.

What is the optimal concentration for using GRPR-FITC antibodies in different applications?

The optimal concentration for GRPR-FITC antibodies varies by application and specific product. While exact optimal concentrations should be determined empirically for each experimental system, here are general guidelines based on the available information:

For ELISA:

• Starting dilution of 1:1000 to 1:5000, optimizing based on signal-to-noise ratio

For Immunofluorescence:

• Typical starting dilutions range from 1:50 to 1:200

• Higher concentrations (1:50) may be needed for tissues with low GRPR expression

For Western Blotting:

• Starting dilutions of 1:1000, adjusting based on band intensity and background

When working with FITC-conjugated antibodies, it's important to remember that the fluorophore can photobleach. Therefore, minimize exposure to light during incubation steps and optimize imaging parameters to capture data before significant signal degradation occurs.

Always perform a titration experiment (testing multiple concentrations) when using a new antibody or working with a new experimental system to determine the optimal working concentration that provides the best signal-to-noise ratio.

How can I use GRPR-FITC antibodies to investigate the relationship between GRPR and NMBR in itch signaling pathways?

GRPR and NMBR (Neuromedin B receptor) are both members of the mammalian bombesin receptor family with distinct but potentially overlapping roles in itch signaling. Research has shown that they are expressed in largely non-overlapping patterns in the superficial spinal cord, suggesting specific functions in sensory processing . To investigate their relationship using GRPR-FITC antibodies:

• Co-localization studies:

  • Use GRPR-FITC antibodies in combination with NMBR antibodies (conjugated with a different fluorophore) to visualize their distribution patterns in spinal cord sections

  • This approach would allow direct visualization of potential overlap or distinct expression domains

• Functional studies in knockout models:

  • Compare GRPR labeling patterns in wild-type, NMBR knockout, and GRPR knockout mouse models

  • Research has shown that GRPR activity is enhanced in NMBR knockout mice despite the lack of upregulation of GRPR expression, suggesting compensatory mechanisms

• Signaling pathway investigation:

  • Use GRPR-FITC antibodies to track receptor internalization or trafficking following stimulation with either GRP or NMB

  • This could provide insights into how these related receptors might compensate for each other's function

• Neuronal circuit tracing:

  • Combined with neuroanatomical tracing methods, GRPR-FITC antibodies can help identify how GRPR+ neurons might act downstream of NMBR+ neurons to integrate itch information

This approach could help clarify the findings that "NMBR and GRPR could compensate for the loss of each other to maintain normal histamine-evoked itch, whereas GRPR is exclusively required for chloroquine-evoked scratching behavior" .

What methodological considerations should be taken when using GRPR-FITC antibodies in multi-color immunofluorescence studies?

When designing multi-color immunofluorescence studies incorporating GRPR-FITC antibodies, several methodological considerations are critical for obtaining reliable results:

• Spectral compatibility:

  • FITC emits green fluorescence (peak emission ~520 nm)

  • Choose companion fluorophores with minimal spectral overlap (e.g., Cy3, Cy5, or Alexa Fluor 594/647)

  • When using multiple fluorophores, sequential imaging rather than simultaneous acquisition may reduce crossover

• Antibody compatibility:

  • If using multiple primary antibodies, ensure they are raised in different host species to avoid cross-reactivity

  • For example, if using rabbit anti-GRPR-FITC, select mouse or goat antibodies for other targets

• Fixation and antigen retrieval optimization:

  • Different antibodies may require different fixation protocols (PFA, methanol, etc.)

  • Test whether the FITC conjugation affects the antibody's performance under various fixation conditions

  • Some epitopes may require antigen retrieval methods that could affect FITC fluorescence

• Signal amplification considerations:

  • Direct FITC conjugation provides a single fluorophore per antibody molecule

  • For low-abundance targets, consider whether secondary amplification methods might be needed

  • Note that additional amplification steps introduce complexity and potential background

• Controls for multi-color studies:

  • Single-stain controls for each fluorophore to establish proper compensation settings

  • Fluorescence minus one (FMO) controls to accurately identify positive populations

  • Secondary-only controls for each fluorophore used

• Photobleaching prevention:

  • FITC is somewhat prone to photobleaching

  • Use anti-fade mounting media with appropriate preservatives

  • Capture FITC images first in sequential imaging protocols

How do post-translational modifications of GRPR affect antibody recognition, and how can this impact experimental interpretation?

Post-translational modifications (PTMs) of GRPR can significantly affect antibody recognition and experimental outcomes when using GRPR-FITC antibodies. Understanding these effects is crucial for accurate interpretation of results:

• Phosphorylation effects:

  • GRPR, like many G-protein coupled receptors, undergoes regulatory phosphorylation after agonist binding

  • Phosphorylation at specific serine/threonine residues may mask antibody epitopes, particularly if the antibody targets regions containing these sites

  • Antibodies targeting the N-terminal region (like some FITC-conjugated versions) may be less affected by activation-dependent phosphorylation

• Glycosylation considerations:

  • GRPR contains potential N-glycosylation sites in its extracellular domains

  • Different tissue or cell types may exhibit different glycosylation patterns

  • This can affect antibody binding if the glycan structures interfere with epitope accessibility

  • When comparing GRPR detection across tissue types, consider that differences in staining intensity might reflect glycosylation differences rather than expression levels

• Receptor internalization and trafficking:

  • Upon activation, GRPR undergoes internalization

  • This changes the subcellular localization and potentially the conformation of the receptor

  • Antibodies may have differential access to epitopes depending on receptor localization

  • Consider using membrane permeabilization protocols appropriate for detecting internalized receptors

• Experimental recommendations:

  • Use multiple antibodies targeting different epitopes when possible

  • Compare results from antibodies targeting extracellular versus intracellular domains

  • Consider treating samples with deglycosylating enzymes to normalize detection if glycosylation is affecting results

  • Document the specific peptide sequence used as immunogen when reporting results, as this clarifies which region of GRPR the antibody recognizes

What approaches can be used to validate the specificity of GRPR-FITC antibodies in neuronal tissue samples?

Validating the specificity of GRPR-FITC antibodies in neuronal tissue is crucial for reliable interpretation of experimental results. Here are comprehensive approaches for antibody validation:

• Genetic validation approaches:

  • Compare staining in wild-type versus GRPR knockout tissues

  • This is the gold standard for antibody validation

  • Research has used GRPR knockout mice to establish specificity of signaling

  • Decreased or absent staining in knockout tissue provides strong evidence of specificity

• Peptide blocking experiments:

  • Pre-incubate the GRPR-FITC antibody with excess immunizing peptide

  • This should competitively inhibit specific binding

  • Use the specific blocking peptide recommended for the antibody (e.g., Catalog # AAP76226 for certain anti-GRPR antibodies)

  • Compare staining patterns with and without peptide blocking

• RNA-protein correlation:

  • Perform in situ hybridization for GRPR mRNA in adjacent tissue sections

  • Compare the pattern with GRPR-FITC antibody immunofluorescence

  • Concordant patterns suggest antibody specificity

  • This is particularly valuable in neuronal tissues where cellular heterogeneity is high

• Multiple antibody validation:

  • Use multiple antibodies targeting different epitopes of GRPR

  • Consistent staining patterns across antibodies suggest specificity

  • Compare antibodies recognizing the N-terminal region versus other domains

• Heterologous expression systems:

  • Test antibody on cells with controlled expression of GRPR (overexpression or inducible systems)

  • Include related receptors (e.g., NMBR) to test for cross-reactivity

  • Western blot analysis showing a single band of appropriate molecular weight

• Functional correlation:

  • Correlate antibody staining with functional responses to GRPR agonists

  • Areas with higher GRPR immunoreactivity should show stronger responses to GRP

  • This approach connects anatomical detection with physiological function

How can I troubleshoot weak or absent signal when using GRPR-FITC antibodies in immunofluorescence?

When encountering weak or absent signal with GRPR-FITC antibodies in immunofluorescence experiments, consider these systematic troubleshooting approaches:

• Antibody-related factors:

  • Verify antibody viability: Excessive freeze-thaw cycles or improper storage may have damaged the antibody or caused photobleaching of the FITC conjugate

  • Check concentration: Increase antibody concentration incrementally to determine if signal improves

  • Confirm lot performance: Test a different lot or request validation data from the manufacturer

• Sample preparation issues:

  • Fixation optimization: Overfixation can mask epitopes; try different fixation protocols or durations

  • Antigen retrieval: Try various methods (heat-induced, enzymatic) to improve epitope accessibility

  • Permeabilization: Ensure adequate membrane permeabilization for accessing intracellular epitopes

  • Blocking optimization: Test different blocking reagents to reduce background while preserving specific signal

• Detection system considerations:

  • Microscope settings: Ensure proper excitation/emission filter sets for FITC (excitation ~495 nm, emission ~520 nm)

  • Signal amplification: Consider switching to a non-conjugated primary with amplified secondary detection system

  • Photobleaching: Minimize sample exposure to light during processing and use anti-fade mounting medium

• Biological considerations:

  • Expression level: Confirm that your sample type expresses detectable levels of GRPR

  • Receptor localization: GRPR distribution may be punctate or localized to specific subcellular compartments

  • Developmental or physiological regulation: Check if GRPR expression is age, activity, or state-dependent

• Positive controls:

  • Include samples known to express high levels of GRPR

  • Consider using transfected cells overexpressing GRPR as a positive control

What strategies can be employed to reduce background fluorescence when using GRPR-FITC antibodies?

High background is a common challenge when working with fluorescently conjugated antibodies. Here are specific strategies to reduce background when using GRPR-FITC antibodies:

• Optimizing blocking conditions:

  • Use species-appropriate serum (5-10%) in blocking buffer

  • Try alternative blocking agents such as BSA, casein, or commercial blocking buffers

  • Extend blocking time to 1-2 hours at room temperature or overnight at 4°C

  • Add 0.1-0.3% Triton X-100 or 0.05% Tween-20 to blocking buffer to reduce non-specific binding

• Antibody dilution and incubation optimization:

  • Test a range of antibody dilutions to find optimal signal-to-noise ratio

  • Extend primary antibody incubation time at 4°C (overnight or longer)

  • Always dilute the GRPR-FITC antibody in blocking buffer

  • Include 0.05-0.1% detergent in antibody dilution buffer

• Washing steps enhancement:

  • Increase number and duration of washes (5-6 washes, 10 minutes each)

  • Use PBS-T (PBS with 0.1% Tween-20) for more stringent washing

  • Perform washing steps with gentle agitation

• Tissue/sample-specific considerations:

  • For tissues with high autofluorescence (e.g., brain), try treating sections with Sudan Black B (0.1-0.3% in 70% ethanol) for 10 minutes after antibody incubation

  • Consider using TrueBlack® or similar reagents specifically designed to reduce autofluorescence

  • For fixed cells, shorter fixation times may reduce background

• Fluorophore considerations:

  • If FITC background is problematic due to sample autofluorescence in the green channel, consider requesting an alternative conjugate (e.g., Cy3, Alexa Fluor 647)

  • Use mounting media with anti-fade agents specifically formulated for FITC

• Controls for identifying sources of background:

  • Include a negative control sample with the FITC-conjugated isotype control antibody

  • This helps distinguish between non-specific antibody binding and tissue autofluorescence

How can I determine if cross-reactivity with other bombesin receptor family members is affecting my GRPR-FITC antibody results?

The bombesin receptor family includes GRPR, NMBR (Neuromedin B Receptor), and BRS-3 (Bombesin Receptor Subtype 3), which share sequence homology. Cross-reactivity could potentially affect experimental interpretation. Here's how to assess and address this issue:

• Sequence analysis and epitope mapping:

  • Review the immunogen sequence used to generate the GRPR-FITC antibody

  • Compare this sequence with corresponding regions in NMBR and BRS-3 using alignment tools

  • Antibodies targeting the N-terminal region of GRPR may have lower cross-reactivity as this region tends to be less conserved among family members

  • The synthetic peptide sequence provided for some antibodies (e.g., FLLNLEVDHFMHCNISSHSADLPVNDDWSHPGILYVIPAVYGVIILIGLI) can be compared with other receptor sequences

• Experimental validation approaches:

  • Test the antibody on cells expressing only NMBR or BRS-3

  • Use receptor-specific knockout tissues or cells:

    • Compare staining in wild-type, GRPR KO, NMBR KO, and double knockout (DKO) samples

    • If staining persists in GRPR KO samples, cross-reactivity is likely

• Competitive binding assays:

  • Pre-incubate the antibody with peptides derived from each receptor type

  • If peptides from non-target receptors block binding, cross-reactivity is indicated

• Differential expression analysis:

  • Leverage the distinct expression patterns of these receptors in different tissues

  • GRPR and NMBR are expressed in largely non-overlapping patterns in the spinal cord

  • Compare antibody staining patterns with known expression patterns from in situ hybridization data

• Receptor-specific functional correlations:

  • GRPR and NMBR mediate distinct aspects of itch signaling

  • Correlate GRPR-FITC staining with functional responses specific to GRP versus NMB stimulation

  • In knockout models, GRPR is specifically required for chloroquine-evoked scratching behavior, while both NMBR and GRPR contribute to histamine-evoked itch

How should I quantify and compare GRPR expression levels across different experimental conditions using GRPR-FITC antibodies?

Accurate quantification of GRPR expression using FITC-conjugated antibodies requires standardized approaches to enable valid comparisons across experimental conditions:

• Immunofluorescence quantification methods:

  • Measure mean fluorescence intensity (MFI) within defined regions of interest (ROIs)

  • Count positive cells using appropriate thresholding

  • Assess subcellular distribution patterns (membrane vs. cytoplasmic localization)

  • Use specialized software (ImageJ, CellProfiler, etc.) for unbiased analysis

• Flow cytometry approaches:

  • Quantify GRPR-FITC signal as mean or median fluorescence intensity

  • Use standardized gating strategies based on appropriate controls

  • Express results as fold change relative to control samples or as molecules of equivalent soluble fluorochrome (MESF)

• Standardization and normalization:

  • Include calibration beads with known fluorescence intensities in each experiment

  • Use internal reference standards (housekeeping proteins) for normalization

  • Process and image all samples in parallel using identical settings

  • For multi-day experiments, include a standard sample in each batch

• Statistical analysis recommendations:

  • Apply appropriate statistical tests based on data distribution

  • Use multiple biological and technical replicates (minimum n=3)

  • Report effect sizes along with p-values

  • Consider using ANOVA with post-hoc tests for multiple condition comparisons

• Reporting standards:

  • Document all image acquisition parameters (exposure time, gain, binning)

  • Report antibody details including catalog number, lot, dilution

  • Specify exact quantification methodology including software and parameters

  • Include representative images alongside quantitative data

Using these standardized approaches allows for reliable comparison of GRPR expression across different experimental conditions, whether examining changes in disease models, after pharmacological interventions, or in different cell populations.

What are the key considerations when interpreting changes in GRPR localization versus expression level in imaging studies?

Distinguishing between changes in GRPR localization and actual expression levels is crucial for accurate interpretation of imaging studies using GRPR-FITC antibodies:

Understanding these considerations helps researchers accurately interpret whether observed changes reflect true alterations in GRPR expression or represent redistribution of the existing receptor pool in response to experimental manipulation.

How do GRPR-FITC antibodies compare with other detection methods for studying GRPR in neuronal circuits?

Different detection methods offer complementary approaches to studying GRPR in neuronal circuits, each with distinct advantages and limitations:

For comprehensive neuronal circuit analysis, combining GRPR-FITC immunolabeling with functional approaches provides the most complete picture. For example, researchers studying itch circuits have effectively combined immunohistochemistry with knockout models to understand how GRPR and NMBR interact in itch signaling pathways . This revealed that "GRPR+ neurons are likely to act downstream of NMBR+ neurons to integrate NMB-NMBR-encoded histaminergic itch information in normal physiological conditions" .

When designing experiments, consider using GRPR-FITC antibodies for precise anatomical localization, followed by functional validation through either calcium imaging or behavioral studies in models with manipulation of GRPR+ neurons.

What are the advantages and limitations of using FITC as a conjugate compared to other fluorophores for GRPR antibodies?

The choice of fluorophore conjugate significantly impacts experimental outcomes when working with GRPR antibodies. Here's a comparative analysis of FITC versus other common fluorophores:

Recommendations based on experimental needs:

• For standard tissue immunofluorescence:

  • FITC is adequate for basic localization

  • Consider Alexa Fluor 488 for improved photostability in the same spectral range

• For multi-label co-localization studies:

  • Pair GRPR-FITC with red/far-red fluorophores (Cy3, Alexa 594/647) for clear spectral separation

  • For three or more labels, consider Alexa Fluor conjugates for better spectral discrimination

• For tissues with high autofluorescence (brain, spinal cord):

  • Avoid FITC in favor of red/far-red fluorophores

  • Alexa Fluor 647 provides optimal signal-to-noise in autofluorescent tissues

• For quantitative analysis of expression levels:

  • Alexa Fluor conjugates provide more stable and consistent signal for quantification

  • For flow cytometry, PE or APC conjugates offer superior sensitivity

When selecting a GRPR antibody conjugate, consider both the experimental requirements and tissue characteristics to optimize signal detection while minimizing background and artifacts.