PREX1 Antibody, FITC conjugated

What is PREX1 and what cellular functions does it regulate?

PREX1 (Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1) functions as a RAC guanine nucleotide exchange factor (GEF), which activates Rac proteins by exchanging bound GDP for free GTP. Its activity is synergistically activated by phosphatidylinositol 3,4,5-trisphosphate and the beta-gamma subunits of heterotrimeric G proteins . PREX1 likely functions downstream of heterotrimeric G proteins, particularly in neutrophils, and plays crucial roles in cell migration, cytoskeletal reorganization, and immune cell function.

PREX1 is predominantly expressed in peripheral blood leukocytes and brain tissue, with intermediate expression in spleen and lymph nodes, and lower expression in other tissues . The functional significance of this tissue-specific expression pattern relates to PREX1's role in immune cell signaling and potential neuronal functions.

What applications are FITC-conjugated PREX1 antibodies suitable for?

FITC-conjugated PREX1 antibodies can be used in multiple experimental applications, including:

• ELISA: Recommended dilution ranges from 1:16,000 to 1:20,000-1:50,000

• Western Blot: Effective dilutions typically between 1:100-500 or 1:500-1:750

• Immunohistochemistry (IHC): Optimal at 1:50-1:150

• Immunoprecipitation (IP): Best results at approximately 1:200

• Flow cytometry/FLISA: Recommended dilution of 1:1000

These applications leverage the fluorescent properties of the FITC conjugate, which eliminates the need for secondary antibody incubation in fluorescence-based detection methods, simplifying experimental workflows and reducing background issues.

What are the optimal storage conditions for FITC-conjugated PREX1 antibodies?

For long-term storage, FITC-conjugated PREX1 antibodies should be kept at -20°C . The antibody is typically shipped at 4°C and upon delivery should be aliquoted to avoid repeated freeze-thaw cycles which can compromise antibody integrity and fluorophore activity . Some suppliers explicitly warn "Do not freeze!" for certain formulations , indicating that specific product guidelines should always be followed.

The antibody is generally supplied in stabilization buffers (0.6 μg/μl concentration) or in Tris Buffered Saline (pH 7.3) containing 0.5% BSA and 0.02% Sodium Azide . These formulations help maintain antibody stability and functionality during storage.

What are the reactivity profiles of available PREX1 antibodies?

Different PREX1 antibodies show varying species reactivity profiles:

When selecting an antibody for cross-species applications, it's critical to verify the specific epitope sequence and consider the degree of conservation across species to ensure reliable results.

What controls should be included when validating FITC-conjugated PREX1 antibodies?

When validating FITC-conjugated PREX1 antibodies for research applications, several controls are essential:

• Positive controls: Cell lines or tissues with known PREX1 expression (peripheral blood leukocytes, brain tissue, or spleen samples)

• Negative controls:

  • Primary antibody omission

  • Isotype controls (e.g., FITC-conjugated rabbit IgG for rabbit polyclonal antibodies)

  • PREX1 knockout or knockdown samples (if available)

• Peptide competition/blocking: Pre-incubating the antibody with the immunizing peptide (e.g., the synthetic peptide corresponding to amino acid region 815-836 of Human PREX1)

• Fluorescence controls: Unstained samples to establish autofluorescence baseline

Additionally, when performing multiplexed experiments, proper compensation controls should be included to account for spectral overlap between FITC and other fluorophores.

How can researchers optimize immunohistochemistry protocols using FITC-conjugated PREX1 antibodies?

Optimizing IHC protocols with FITC-conjugated PREX1 antibodies requires careful attention to several parameters:

• Fixation: Optimize fixation conditions (typically 4% paraformaldehyde) to preserve both antigen accessibility and tissue morphology

• Antigen retrieval: Test different antigen retrieval methods (heat-induced in citrate buffer pH 6.0 or EDTA buffer pH 9.0) to maximize epitope exposure

• Blocking: Use 5-10% normal serum from the same species as the secondary antibody plus 0.3% Triton X-100 for permeabilization

• Antibody dilution: Begin with the recommended dilution range (1:50-1:150) and optimize through a dilution series

• Incubation conditions: Test both overnight incubation at 4°C and shorter incubations (2-4 hours) at room temperature

• Counterstaining: Use DAPI for nuclear visualization while ensuring minimal spectral overlap with FITC

• Mounting medium: Use anti-fade mounting medium specifically formulated for fluorescence preservation

• Signal amplification: Consider tyramide signal amplification for low-abundance targets

Remember that FITC is sensitive to photobleaching, so minimize exposure to light during all steps and consider using alternative more photostable fluorophores for long-term imaging studies.

What methodologies allow for quantitative assessment of PREX1 activation states?

Quantifying PREX1 activation states requires sophisticated approaches beyond simple expression analysis:

• Active GEF pull-down assays: Using GST-Rac1 (nucleotide-free) as bait to selectively capture active PREX1

• Phospho-specific antibody analysis: Detecting phosphorylation at key regulatory sites that correlate with PREX1 activation

• FRET-based biosensors: Developing FRET sensors that respond to PREX1-mediated Rac activation

• Rac-GTP pull-down assays: Indirectly measuring PREX1 activity by quantifying active Rac levels

• Live-cell imaging: Using fluorescently-tagged PREX1 constructs to monitor translocation to membranes upon activation

When using FITC-conjugated PREX1 antibodies in these contexts, researchers should be aware that the fluorophore conjugation might affect antibody binding to certain conformational epitopes that change during PREX1 activation cycles.

How do experimental conditions impact PREX1 detection with FITC-conjugated antibodies?

Several experimental conditions can significantly impact the detection of PREX1 using FITC-conjugated antibodies:

• pH sensitivity: FITC fluorescence is pH-dependent, with optimal emission at slightly alkaline pH (7.5-8.5). Significant pH shifts in buffers can alter signal intensity

• Sample preparation: The effectiveness of detection varies based on sample processing:

  • For Western blotting: Complete protein denaturation may expose epitopes normally hidden in the native conformation

  • For flow cytometry: Fixation and permeabilization protocols significantly impact intracellular epitope accessibility

• Protein interactions: PREX1 interacts with multiple binding partners (PI3K, G-protein βγ subunits), which may mask antibody epitopes in certain contexts

• Post-translational modifications: Phosphorylation states of PREX1 may alter antibody recognition, particularly if the epitope region (e.g., amino acids 815-836) contains modification sites

• Expression levels: PREX1 detection sensitivity varies across tissues, with highest expression in peripheral blood leukocytes and brain, intermediate in spleen and lymph nodes, and weak in other tissues

Researchers should consider these factors when designing experiments and interpreting results, particularly when comparing PREX1 detection across different experimental conditions.

What strategies can address potential pitfalls in multiplexed experiments using FITC-PREX1 antibodies?

Multiplexed experiments combining FITC-conjugated PREX1 antibodies with other fluorescent probes present several challenges:

• Spectral overlap: FITC emission overlaps with several common fluorophores. Implement proper spectral compensation and consider:

  • Sequential scanning in confocal microscopy

  • Careful fluorophore selection (e.g., pairing FITC with far-red dyes rather than PE or TRITC)

  • Linear unmixing algorithms for highly multiplexed experiments

• Channel bleed-through: Minimize by:

  • Optimizing antibody concentrations to use the minimum required for specific detection

  • Using narrow bandpass filters for detection

  • Acquiring single-stained controls for compensation matrices

• Differential photostability: FITC bleaches more rapidly than many other fluorophores. Address by:

  • Imaging FITC channels first in sequential acquisition

  • Using anti-fade reagents specifically optimized for FITC

  • Considering photoconversion of FITC to more stable derivatives

• Cross-reactivity issues: When using multiple primary antibodies:

  • Perform careful blocking with species-specific sera

  • Consider using antibody fragments or directly conjugated primaries

  • Validate staining patterns with single-stain controls

• Quantification challenges: For accurate quantitative multiplexed analysis:

  • Establish standard curves for each fluorophore

  • Account for differential quantum yields

  • Use appropriate normalization strategies for comparative analysis

How can PREX1 antibodies contribute to understanding the role of PREX1 in disease pathways?

FITC-conjugated PREX1 antibodies can significantly advance our understanding of PREX1's role in various disease processes:

• Cancer research: PREX1 is implicated in cancer cell migration and metastasis. FITC-conjugated antibodies enable:

  • Visualization of PREX1 localization during cell migration

  • Quantification of expression levels across different tumor types

  • Correlation of PREX1 expression with disease progression markers

• Immune disorders: Given PREX1's high expression in leukocytes , these antibodies facilitate:

  • Analysis of PREX1 dynamics during immune cell activation

  • Assessment of PREX1 dysregulation in autoimmune conditions

  • Evaluation of PREX1's role in leukocyte migration and function

• Neurodegenerative diseases: With significant PREX1 expression in brain tissue , researchers can:

  • Map PREX1 distribution across different brain regions

  • Investigate changes in PREX1 levels or localization in disease models

  • Explore PREX1's role in neuronal signaling and plasticity

• Therapeutic target validation: FITC-conjugated antibodies support:

  • High-content screening approaches to identify PREX1 modulators

  • Validation of target engagement by potential therapeutics

  • Monitoring of PREX1 expression/activation following experimental treatments