PREX2 Antibody, FITC conjugated

What is PREX2 and why is it significant in cancer research?

PREX2 (Phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 2, also known as P-Rex2a) is an 182 kDa protein that functions as a RAC1 guanine nucleotide exchange factor (GEF), activating Rac proteins by exchanging bound GDP for free GTP. PREX2 is sensitive to phosphoinositide 3-kinase (PI3K) and belongs to the Rac guanine nucleotide exchange factor family (RacGEFs). Its activity is synergistically activated by phosphatidylinositol 3,4,5-trisphosphate and the beta gamma subunits of heterotrimeric G protein .

PREX2 has emerged as a significant factor in cancer research because it has been identified as an oncogene with abnormal expression in various tumors including melanoma, breast, ovarian, prostatic, hepatocellular carcinoma, and pancreatic cancer. Recent research has also demonstrated that PREX2 contributes significantly to radiation resistance in colorectal cancer by inhibiting radiotherapy-induced tumor immunogenicity via the cGAS/STING/IFNs pathway .

What is FITC conjugation and why is it used with antibodies?

FITC (Fluorescein isothiocyanate) conjugation is a process where FITC, a green fluorescent dye with an excitation wavelength of 495nm and emission wavelength of 519nm, is chemically attached to antibodies or other proteins. The conjugation occurs through the reaction of the isothiocyanate group of FITC with primary amines (mainly lysine residues) on the antibody .

FITC conjugation is used with antibodies to enable visualization of target proteins in techniques such as immunofluorescence microscopy, flow cytometry, and immunohistochemistry. Once conjugated to an antibody, FITC serves as a reporter molecule that emits fluorescence when excited by light of the appropriate wavelength, allowing researchers to detect and localize specific antigens in biological samples without the need for secondary antibody detection systems .

What are the basic buffer requirements for successful FITC conjugation to PREX2 antibodies?

For successful FITC conjugation to any antibody, including PREX2 antibodies, the antibody should ideally be in an amine-free buffer with pH ranging from 6.5 to 8.5, and at a concentration of 0.5-5 mg/ml. The presence of amine-containing buffers (such as Tris or glycine) can interfere with the conjugation reaction by competing with the antibody amines for reaction with FITC .

If the antibody is not in a compatible buffer, it should be purified and buffer-exchanged before conjugation. Common compatible buffers include phosphate-buffered saline (PBS), HEPES, and sodium carbonate. Additionally, the antibody solution should be free of carriers like BSA or gelatin, as these proteins also contain amine groups and would become labeled with FITC, reducing the efficiency of antibody labeling .

What applications are FITC-conjugated PREX2 antibodies commonly used for?

FITC-conjugated PREX2 antibodies can be used in various research applications, primarily:

• Immunofluorescence microscopy to visualize PREX2 localization in fixed cells or tissue sections

• Flow cytometry for detecting and quantifying PREX2 expression in cell populations

• Immunohistochemistry for examining PREX2 expression in tissue specimens

• Tracking PREX2 expression changes in response to treatments or genetic manipulations

These applications are particularly valuable in cancer research, where PREX2 has been identified as a potential biomarker for radioresistance and a therapeutic target, especially in colorectal cancer. Researchers can use FITC-conjugated PREX2 antibodies to monitor changes in PREX2 expression or localization following treatments such as ionizing radiation or PREX2 inhibitors like PREX-in1 .

How does PREX2 contribute to radiation resistance in colorectal cancer and how can FITC-conjugated antibodies help study this mechanism?

PREX2 contributes to radiation resistance in colorectal cancer through multiple mechanisms revealed by recent research:

• DNA repair enhancement: PREX2 facilitates DNA repair by upregulating DNA-PKcs, helping cancer cells recover from radiation-induced DNA damage

• Suppression of immunogenic cell death: PREX2 inhibits radiation-induced immunogenic cell death markers

• Inhibition of CD8+ T cell infiltration: PREX2 impedes anti-tumor immune responses by blocking the cGAS/STING/IFNs pathway

FITC-conjugated PREX2 antibodies can help study these mechanisms by enabling:

• Visualization of PREX2 localization during DNA damage response using immunofluorescence microscopy

• Quantification of PREX2 expression levels before and after radiation treatment using flow cytometry

• Co-localization studies with DNA repair proteins or components of the cGAS/STING pathway

• Tracking PREX2 expression changes in response to radiation therapy or PREX2 inhibitors

These applications allow researchers to better understand the spatiotemporal dynamics of PREX2 in radiation resistance and develop strategies to overcome this resistance by targeting PREX2 .

What are the optimal conjugation conditions to achieve the highest quality FITC-labeled PREX2 antibodies?

Achieving high-quality FITC-labeled PREX2 antibodies requires optimization of several parameters:

• Antibody purity: Use relatively pure IgG, preferably obtained by DEAE Sephadex chromatography, as contaminant proteins will also become labeled

• FITC quality: Use high-quality FITC to ensure consistent labeling and minimize batch variation

• Reaction conditions:

  • pH: Optimal conjugation occurs at pH 9.5

  • Temperature: Room temperature (20-25°C) provides efficient conjugation

  • Protein concentration: An initial concentration of 25 mg/ml yields maximal labeling

  • Reaction time: Maximal labeling is typically achieved within 30-60 minutes

After conjugation, it's important to separate optimally labeled antibodies from under- and over-labeled proteins using gradient DEAE Sephadex chromatography. This separation helps achieve a consistent fluorescein/protein (F/P) ratio, which is critical for reproducible experimental results. The F/P ratio affects both the fluorescence intensity and the antibody's binding capacity, with excessively high F/P ratios potentially compromising antibody function .

How can researchers optimize PREX2 detection in flow cytometry when using FITC-conjugated antibodies?

Optimizing PREX2 detection in flow cytometry with FITC-conjugated antibodies requires attention to several technical aspects:

• Permeabilization protocol: Since PREX2 is primarily intracellular, effective permeabilization is crucial. For flow cytometry, researchers should optimize permeabilization using agents like Triton X-100 (0.1%) for fixed cells or saponin-based buffers for gentler permeabilization .

• Antibody titration: Determine the optimal concentration of FITC-conjugated PREX2 antibody by testing a range of dilutions. This prevents both insufficient signal and excessive background.

• Signal amplification: For low-abundance targets like PREX2, consider using biotinylated primary antibodies with FITC-streptavidin for signal amplification.

• Compensation controls: Since FITC emission can spill into other channels, proper compensation controls should be used, especially in multicolor flow cytometry panels.

• Negative controls: Include isotype controls conjugated with FITC at the same concentration to distinguish non-specific binding from true PREX2 signal .

• Cell fixation: If performing intracellular PREX2 staining, optimize fixation conditions (typically 4% paraformaldehyde for 10 minutes) to preserve cellular structure while maintaining PREX2 epitope accessibility .

What are the key considerations when using FITC-conjugated PREX2 antibodies to study the cGAS/STING/IFNs pathway in cancer immunotherapy research?

When using FITC-conjugated PREX2 antibodies to study the cGAS/STING/IFNs pathway in cancer immunotherapy research, researchers should consider:

• Multiplexed immunofluorescence: Combine FITC-conjugated PREX2 antibodies with antibodies against cGAS, STING, and downstream effectors (labeled with different fluorophores) to visualize pathway relationships. This requires careful selection of compatible fluorophores and proper control of spectral overlap.

• Functional readouts: Correlate PREX2 expression (detected by FITC-conjugated antibodies) with functional markers of the cGAS/STING pathway, such as:

  • Type I interferon production

  • HMGB1 release (measurable by ELISA)

  • Calreticulin expression

  • Extracellular ATP levels

• Radiation response studies: Design experiments that correlate PREX2 levels (using FITC-conjugated antibodies) with radiation-induced activation of the cGAS/STING pathway. This should include time-course analyses to capture dynamic changes.

• Tumor microenvironment analysis: Assess how PREX2 expression impacts immune cell infiltration by combining FITC-conjugated PREX2 antibody staining with markers for CD8+ T cells and other immune populations in tissue sections or by flow cytometry of tumor-infiltrating lymphocytes .

• Inhibitor studies: Use PREX2 inhibitors like PREX-in1 to modulate PREX2 function and monitor consequent changes in cGAS/STING pathway activation and immune cell recruitment .

What is the step-by-step protocol for conjugating FITC to PREX2 antibodies?

The following is a detailed protocol for conjugating FITC to PREX2 or other antibodies:

• Antibody preparation:

  • Ensure the antibody is in a compatible buffer (10-50mM amine-free buffer, pH 6.5-8.5)

  • Adjust antibody concentration to 1-5 mg/ml

  • Remove any amine-containing compounds through buffer exchange if necessary

• Conjugation procedure:

  • Add 1 μl of Modifier reagent to each 10 μl of antibody solution and mix gently

  • Remove the cap from the vial of FITC Mix (lyophilized material)

  • Pipette the antibody sample with added Modifier directly onto the lyophilized FITC

  • Resuspend gently by withdrawing and re-dispensing the liquid once or twice

  • Replace the cap and incubate in the dark at room temperature (20-25°C) for 3 hours

  • For optimal results at higher pH, use 0.1M carbonate buffer at pH 9.5 and incubate for 30-60 minutes

• Quenching and purification:

  • After incubation, add 1 μl of Quencher reagent for each 10 μl of antibody used

  • For larger scale preparations, purify the conjugate using gradient DEAE Sephadex chromatography to separate optimally labeled antibodies from under- and over-labeled proteins

• Storage:

  • Store the conjugated antibody at 4°C in the dark (short-term)

  • For long-term storage, add appropriate stabilizers and store at -20°C in small aliquots to avoid freeze-thaw cycles

How can researchers troubleshoot common issues with FITC-conjugated PREX2 antibody staining in immunofluorescence?

Here are solutions to common problems encountered when using FITC-conjugated PREX2 antibodies in immunofluorescence:

• Weak or no signal:

  • Increase antibody concentration

  • Optimize fixation method (try 4% paraformaldehyde for 10 minutes)

  • Improve permeabilization (try 0.1% Triton X-100 for approximately 30 minutes)

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

  • Try antigen retrieval methods if using tissue sections

  • Check if the F/P ratio is optimal (too low or too high can reduce signal)

• High background or non-specific staining:

  • Increase blocking time (use 1% BSA solution for at least 30 minutes)

  • Reduce antibody concentration

  • Include additional blocking agents (normal serum from the same species as secondary antibody)

  • Add 0.1-0.3% Triton X-100 to blocking and antibody solutions

  • Ensure all washing steps are thorough (at least 3x5 minutes)

• Photobleaching of FITC signal:

  • Use anti-fade mounting medium

  • Minimize exposure to light during sample preparation

  • Capture images quickly or use anti-photobleaching agents

  • Consider using more photostable fluorophores for critical applications

• Autofluorescence interference:

  • Include an unstained control to identify sources of autofluorescence

  • Use Sudan Black B (0.1-0.3%) to reduce autofluorescence

  • Consider spectral unmixing during image acquisition

What buffer compatibility issues should researchers be aware of when working with FITC conjugation to PREX2 antibodies?

Researchers should be aware of several buffer compatibility issues when performing FITC conjugation to PREX2 or other antibodies:

• Incompatible buffers that interfere with conjugation:

  • Amine-containing buffers (Tris, glycine, ammonium salts) compete with antibody amines for reaction with FITC

  • Buffers with primary amines or strong nucleophiles

  • Carrier proteins like BSA or gelatin (contain amine groups)

  • High concentrations of sodium azide (>0.1%)

• Compatible buffers:

  • Phosphate-buffered saline (PBS)

  • HEPES buffer

  • Sodium carbonate buffer (particularly effective at pH 9.5)

  • MES buffer

  • Sodium borate buffer

• pH considerations:

  • Optimal pH range is 7.5-9.5, with best results at pH 9.5

  • Lower pH (<6.5) significantly reduces conjugation efficiency

  • Higher pH (>9.5) may decrease antibody stability

• Salt and stabilizer effects:

  • High salt concentrations (>500mM) can reduce conjugation efficiency

  • Some stabilizers can interfere with the reaction

  • Detergents at concentrations >0.1% may affect conjugation

If the antibody is not in a compatible buffer, researchers should perform buffer exchange using dialysis, gel filtration, or concentrator devices with appropriate molecular weight cutoffs before attempting conjugation .

How can researchers validate the specificity and functionality of FITC-conjugated PREX2 antibodies for their experiments?

To validate FITC-conjugated PREX2 antibodies for research use, follow these methodological approaches:

• Western blot validation:

  • Compare staining patterns of unconjugated vs. FITC-conjugated PREX2 antibodies

  • Confirm the antibody detects the expected band size (PREX2 is approximately 183 kDa)

  • Include positive controls (PREX2 transfected cell lysates) and negative controls (non-transfected cell lysates)

  • Verify that conjugation hasn't altered antibody specificity

• Overexpression and knockdown controls:

  • Use PREX2 overexpression systems (e.g., PREX2 cloned into pEZ-Lv201 vector) as positive controls

  • Generate PREX2 knockdown cell lines using shRNA (in pLKD-Puro vector) as negative controls

  • Confirm that antibody signal increases with overexpression and decreases with knockdown

• Fluorescence intensity and F/P ratio determination:

  • Measure the absorbance of the conjugate at 280nm (protein) and 495nm (FITC)

  • Calculate the F/P ratio to ensure optimal labeling (typically 3-8 FITC molecules per antibody)

  • Too high F/P ratio (>8) may indicate over-labeling that could affect antibody function

• Functional assays:

  • Verify that the FITC-conjugated antibody can detect changes in PREX2 expression after relevant treatments (e.g., radiation)

  • Confirm that staining patterns correlate with expected biological processes (e.g., DNA repair after radiation)

• Cross-reactivity testing:

  • Test the antibody on samples known to lack PREX2 expression

  • Use isotype controls conjugated with FITC at the same concentration

What experimental design is recommended for studying PREX2's role in radiation resistance using FITC-conjugated antibodies?

A comprehensive experimental design for studying PREX2's role in radiation resistance using FITC-conjugated antibodies should include:

• Cell line models:

  • Paired radioresistant and radiosensitive colorectal cancer cell lines (e.g., IR-SW480 vs. SW480)

  • PREX2 overexpression models using lentiviral transduction

  • PREX2 knockdown models using shRNA

  • Control cell lines with empty vectors

• Radiation treatment protocol:

  • Define radiation doses (typically 2-8 Gy)

  • Establish time points for analysis (immediate, 24h, 48h, 72h post-radiation)

  • Include non-irradiated controls for each cell line

• FITC-conjugated PREX2 antibody applications:

  • Immunofluorescence to track PREX2 localization before and after radiation

  • Flow cytometry to quantify PREX2 expression changes at single-cell resolution

  • Co-staining with DNA damage markers (γH2AX) and DNA repair proteins

  • Co-staining with immunogenic cell death markers (calreticulin)

• Functional readouts:

  • Colony formation assays to assess survival after radiation

  • Comet assay to measure DNA damage repair

  • Apoptosis assays using Annexin V/PI

  • Immunogenic cell death markers (HMGB1, ATP release, calreticulin exposure)

  • T cell infiltration and activation markers

• In vivo validation:

  • Xenograft mouse models comparing PREX2-high and PREX2-low tumors

  • Radiation treatment protocol

  • Analysis of tumor growth, immune infiltration, and molecular markers

  • Testing PREX2 inhibitors (e.g., PREX-in1) in combination with radiation

How should researchers quantify and interpret FITC-conjugated PREX2 antibody signals in both flow cytometry and immunofluorescence applications?

Proper quantification and interpretation of FITC-conjugated PREX2 antibody signals requires standardized approaches:

• Flow cytometry quantification:

  • Report median fluorescence intensity (MFI) rather than mean to reduce impact of outliers

  • Always subtract autofluorescence using unstained controls

  • Calculate the fold change relative to appropriate controls (e.g., isotype control, untreated sample)

  • For absolute quantification, use calibration beads with known fluorophore molecules

  • When comparing different conditions, normalize to housekeeping proteins or use ratio metrics

• Immunofluorescence quantification:

  • Capture images using consistent exposure settings

  • Measure fluorescence intensity using software like ImageJ

  • Quantify on a per-cell basis using nuclear counterstains to identify individual cells

  • Consider both intensity and subcellular distribution patterns

  • For co-localization studies, use Pearson's correlation coefficient or Manders' overlap coefficient

• Statistical analysis:

  • Use appropriate statistical tests (t-test for two conditions, ANOVA for multiple conditions)

  • Include sufficient biological replicates (minimum n=3)

  • Report both p-values and effect sizes

  • Use non-parametric tests if data do not follow normal distribution

• Interpretation guidelines:

  • Consider the dynamic range of FITC (potential saturation at high expression)

  • Account for potential photobleaching during image acquisition

  • Interpret changes in the context of relevant controls and biological significance

  • For threshold-based analyses, justify threshold selection with appropriate controls

What are the best experimental controls when using FITC-conjugated PREX2 antibodies in radiation resistance studies?

When designing radiation resistance studies using FITC-conjugated PREX2 antibodies, include these essential controls:

• Antibody specificity controls:

  • PREX2 knockdown cells (using validated shRNA constructs)

  • PREX2 overexpression cells (using full-length PREX2 cDNA in expression vectors)

  • Isotype controls conjugated with FITC at the same concentration

  • Blocking peptide controls to confirm epitope specificity

• Conjugation controls:

  • Unconjugated primary antibody followed by FITC-conjugated secondary antibody

  • Different F/P ratio conjugates to determine optimal labeling

  • Different lots of FITC-conjugated antibodies to assess batch variation

• Radiation experiment controls:

  • Non-irradiated samples for each cell line and condition

  • Time-matched controls for each post-radiation time point

  • Positive controls for radiation response (e.g., γH2AX staining)

  • Dose-response curves to determine appropriate radiation doses

• Treatment validation controls:

  • For PREX2 inhibitor studies, include vehicle controls

  • For genetic manipulation, include empty vector controls

  • Positive controls known to modulate radiation sensitivity

  • Functional readouts to confirm radiation response (colony formation, apoptosis assays)

• Imaging and flow cytometry controls:

  • Single-color controls for compensation in multicolor flow cytometry

  • Fluorescence minus one (FMO) controls to set gating boundaries

  • Secondary antibody-only controls to assess non-specific binding

  • Fixed exposure settings and acquisition parameters across experiments

How can FITC-conjugated PREX2 antibodies be incorporated into multiplex immunofluorescence panels for studying tumor microenvironment?

FITC-conjugated PREX2 antibodies can be effectively incorporated into multiplex immunofluorescence panels using these methodological approaches:

• Panel design considerations:

  • FITC works well in multiplex panels due to its bright signal and distinct spectral properties

  • Pair with fluorophores having minimal spectral overlap (e.g., DAPI, Cy3, Cy5, APC)

  • Consider FITC's relatively broad emission spectrum when selecting other fluorophores

  • Limit panel to 4-6 colors for conventional microscopy; more colors may require spectral imaging

• Sequential staining approach:

  • For complex panels, use sequential staining with microwave treatment between rounds

  • Begin with FITC-conjugated PREX2 antibody staining

  • Follow with additional markers like:

    • Cancer cell markers (e.g., cytokeratin)

    • Immune cell markers (CD8, CD4, CD45)

    • Functional markers (cGAS, STING, phospho-STAT1)

• Tyramide signal amplification (TSA) method:

  • For low-abundance targets, enhance FITC signal using TSA

  • This allows for sequential rounds of staining on the same section

  • After each round, heat-inactivate HRP before the next primary antibody application

• Analysis strategies:

  • Use cell segmentation algorithms to identify individual cells

  • Quantify marker co-expression at single-cell resolution

  • Analyze spatial relationships between PREX2+ cells and immune cell populations

  • Correlate PREX2 expression with distance from immune infiltrates

• Validation controls:

  • Single-stain controls for each antibody in the panel

  • Fluorescence minus one (FMO) controls

  • Autofluorescence controls and spectral unmixing when needed

What emerging technologies can enhance FITC-conjugated PREX2 antibody applications in cancer immunotherapy research?

Several emerging technologies can significantly enhance FITC-conjugated PREX2 antibody applications in cancer immunotherapy research:

• Imaging mass cytometry (IMC) or multiplexed ion beam imaging (MIBI):

  • These technologies enable highly multiplexed (30-40+ markers) analysis of tissue sections

  • FITC-conjugated PREX2 antibodies can be integrated into metal-labeled antibody panels

  • Allows simultaneous assessment of PREX2 expression, immune cell phenotypes, and activation states at subcellular resolution

• Spatial transcriptomics combined with protein detection:

  • Technologies like 10x Visium or GeoMx DSP can correlate PREX2 protein expression with spatial gene expression profiles

  • Enables understanding of how PREX2 expression influences the tumor transcriptional landscape

• Live-cell imaging with photoactivatable FITC derivatives:

  • Photoactivatable or photoswitchable FITC variants allow temporal control of fluorescence

  • Enables tracking PREX2 dynamics in living cells during DNA damage response

  • Can be combined with super-resolution microscopy for nanoscale localization

• CRISPR-based screening with FITC readouts:

  • CRISPR screens targeting genes in the PREX2 pathway

  • Use FITC-conjugated PREX2 antibodies as readouts for high-content screening

  • Identify novel regulators of PREX2 expression or function

• Single-cell proteomics approaches:

  • CITE-seq or REAP-seq technologies allow simultaneous measurement of PREX2 and other proteins with transcriptome analysis

  • Microfluidic-based single-cell western blotting with FITC detection

  • Enables correlation of PREX2 levels with cellular states at single-cell resolution

How can researchers develop and validate custom FITC-conjugated PREX2 antibodies for specialized research applications?

Researchers can develop and validate custom FITC-conjugated PREX2 antibodies for specialized applications following these methodological steps:

• Antibody generation:

  • Select appropriate PREX2 epitopes (consider functional domains like the DH domain or regions relevant to specific research questions)

  • Generate monoclonal antibodies through hybridoma technology or recombinant approaches

  • Test antibody specificity against recombinant PREX2 proteins and cell lysates

• FITC conjugation optimization:

  • Determine optimal F/P ratio through controlled conjugation experiments

  • Test different conjugation conditions (pH 7.5-9.5, reaction times 30-180 minutes)

  • Purify conjugates using size exclusion chromatography or ion exchange methods

  • Measure spectroscopic properties to confirm successful conjugation

• Validation in multiple systems:

  • Western blot analysis using positive controls (PREX2 transfected cells) and negative controls

  • Immunoprecipitation to confirm antibody binds native PREX2

  • Immunofluorescence to assess subcellular localization patterns

  • Flow cytometry to confirm detection in single-cell suspensions

• Functional validation:

  • Knockdown experiments using shRNA or CRISPR to confirm signal specificity

  • Overexpression studies to assess detection sensitivity

  • Phosphorylation-specific antibodies to distinguish active vs. inactive PREX2

  • Binding competition assays with unlabeled antibodies

• Application-specific optimization:

  • For flow cytometry: optimize permeabilization protocols

  • For immunohistochemistry: determine optimal antigen retrieval methods

  • For super-resolution microscopy: test with different fixation methods

  • For multiplexed imaging: validate compatibility with other antibodies in the panel

What are the key takeaways for researchers working with FITC-conjugated PREX2 antibodies?

The key takeaways for researchers working with FITC-conjugated PREX2 antibodies include:

• Antibody selection and preparation considerations:

  • Ensure high purity of the antibody before conjugation (IgG purified by DEAE Sephadex chromatography is recommended)

  • Use amine-free buffers at pH 6.5-8.5 for optimal conjugation

  • Optimize antibody concentration (0.5-5mg/ml) and remove carriers like BSA or gelatin

• FITC conjugation optimization:

  • Maximal labeling occurs at pH 9.5, room temperature, with protein concentration around 25 mg/ml

  • Optimal reaction time is typically 30-60 minutes

  • After conjugation, separate optimally labeled antibodies using gradient DEAE Sephadex chromatography

• PREX2 biology insights:

  • PREX2 is a significant oncogene involved in various cancers

  • It contributes to radiation resistance in colorectal cancer by enhancing DNA repair and suppressing immunogenic cell death

  • PREX2 functions through multiple mechanisms including upregulation of DNA-PKcs and inhibition of the cGAS/STING/IFNs pathway

• Experimental design recommendations:

  • Include appropriate controls for antibody specificity, conjugation quality, and biological responses

  • Design multiplexed approaches to study PREX2 in relation to other pathway components

  • Consider both in vitro and in vivo validation when studying PREX2's role in radiation resistance

• Future research directions:

  • PREX2 inhibitors like PREX-in1 show promise for enhancing radiotherapy efficacy

  • Combination approaches targeting PREX2 and immune checkpoints may provide synergistic benefits

  • PREX2 expression may serve as a biomarker for predicting radiotherapy response in colorectal cancer