ALX4 Antibody, FITC conjugated

What is ALX4 and what cellular functions does it regulate?

ALX4 (Homeobox protein aristaless-like 4) is a transcription factor involved in epigenetic regulation and nuclear signaling. This protein, also known as KIAA1788, functions in developmental processes and tissue-specific gene expression. The ALX4 Antibody, FITC conjugated allows researchers to detect and visualize this protein in human samples through various immunological techniques. This antibody specifically targets recombinant Human Homeobox protein aristaless-like 4 protein, particularly the 112-216AA region . For developmental studies, it's essential to understand that ALX4 functions within networks of other transcription factors, making careful experimental design critical when investigating its regulatory roles.

What are the optimal storage conditions for ALX4 Antibody, FITC conjugated?

For ALX4 Antibody, FITC conjugated, optimal storage is critical for maintaining immunoreactivity and fluorescence intensity. Upon receipt, store the antibody at -20°C or -80°C . Avoid repeated freeze-thaw cycles as this can significantly compromise antibody quality and functionality. The antibody is supplied in a storage buffer containing preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) , which help maintain its activity. For FITC-conjugated antibodies in general, protect from exposure to light during storage as continuous light exposure causes gradual loss of fluorescence . For working solutions, store at 4°C in the dark and use within a few days. Some manufacturers specifically warn "Do not freeze!" for certain FITC conjugates , highlighting the importance of checking product-specific storage recommendations.

What are the recommended dilutions for different applications of ALX4 Antibody, FITC conjugated?

The optimal dilution for any antibody application requires balancing signal strength with background. For ALX4 Antibody, FITC conjugated, recommended dilutions vary by application:

What controls should be included when using ALX4 Antibody, FITC conjugated?

Proper controls are essential for validating ALX4 Antibody, FITC conjugated experiments:

• Isotype control: Use a FITC-conjugated rabbit IgG (matching the ALX4 antibody isotype) at the same concentration to assess non-specific binding.

• Negative control: Include unstained samples and samples stained with secondary antibody only (if applicable).

• Positive control: Use cells or tissues known to express ALX4 to confirm antibody performance.

• Blocking control: Pre-incubate the antibody with recombinant ALX4 protein (112-216AA) to confirm specificity.

• Secondary reagent control: When performing flow cytometry, compare with isotype control antibodies to distinguish true signal from background, similar to the methodology shown for other antibodies .

These controls help differentiate between specific binding, non-specific binding, and autofluorescence, particularly important for FITC conjugates which can occasionally yield higher background in certain tissues.

How can ALX4 Antibody, FITC conjugated be used in multi-color immunofluorescence experiments?

Multi-color immunofluorescence requires careful selection of fluorophores to avoid spectral overlap. When using ALX4 Antibody, FITC conjugated in multi-color experiments:

• FITC has excitation/emission peaks at approximately 495/519 nm, making it compatible with common filter sets.

• Pair with fluorophores like Texas Red, Cy3, or Alexa Fluor 594 for red emission, and DAPI or Hoechst for nuclear counterstaining.

• Sequential imaging rather than simultaneous acquisition may be necessary to prevent bleed-through.

• Include single-stained controls for each fluorophore to establish proper compensation settings.

• For co-localization studies with other nuclear proteins, perform z-stack imaging to confirm true co-localization versus overlapping signals from different cellular planes.

When designing these experiments, consider that FITC is more susceptible to photobleaching than some other fluorophores, so incorporate anti-fade reagents and minimize exposure times during image acquisition. For optimal results, image FITC channels first in your acquisition sequence when examining multiple markers .

What are the considerations for using ALX4 Antibody, FITC conjugated in pH-dependent cellular targeting strategies?

pH-dependent cellular targeting strategies can be particularly relevant when using ALX4 Antibody, FITC conjugated, especially in cancer research applications. The inherent acidity of solid tumors can be exploited for selective targeting:

• FITC fluorescence is pH-sensitive, with diminished signal in acidic environments (pH < 6), which must be considered when interpreting results from acidic cellular compartments or tumor microenvironments.

• For targeted studies, consider using pHLIP (pH Low Insertion Peptide) conjugated with FITC as a complementary approach to ALX4 antibody studies, as these peptides selectively insert into membranes under acidic conditions .

• When studying pH-dependent phenomena, include pH calibration controls by treating fixed cells with buffers of known pH to establish a fluorescence-pH correlation curve.

• For experiments involving both ALX4 detection and pH measurement, consider using ratiometric pH indicators alongside FITC-conjugated antibodies.

Research has shown that FITC-conjugated agents can effectively recruit antibodies in a pH-dependent manner, with greater efficacy at tumor-relevant pH (~6.0) compared to physiological pH (7.4) . This property can be leveraged for developing selective immunotherapeutic approaches targeting ALX4-expressing cancer cells.

What sample preparation techniques optimize ALX4 Antibody, FITC conjugated performance in tissue sections?

Successful immunofluorescence with ALX4 Antibody, FITC conjugated in tissue sections requires careful sample preparation:

• Fixation: 4% paraformaldehyde for 10-15 minutes generally preserves both antigenicity and cellular architecture. Avoid over-fixation which can mask epitopes.

• Antigen retrieval: For formalin-fixed tissues, heat-induced epitope retrieval in citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) may be necessary to expose the ALX4 epitope.

• Permeabilization: Use 0.1-0.3% Triton X-100 for nuclear proteins like ALX4, with optimization needed for specific tissue types.

• Blocking: Implement thorough blocking with 10% normal serum (from the same species as the secondary antibody) and 1% BSA to minimize background.

• Antibody incubation: For tissue sections, longer incubation times (overnight at 4°C) at higher dilutions (1:200-1:500) often yield better signal-to-noise ratios than shorter incubations at higher concentrations.

• Washing: Extensive washing with PBS containing 0.05% Tween-20 helps reduce non-specific binding.

For advanced applications like super-resolution microscopy, sample thickness becomes critical. Use thin sections (5-10 μm) and consider tissue clearing techniques if examining thick tissue specimens to improve signal detection of nuclear transcription factors like ALX4.

How does phosphorylation state affect ALX4 detection and what methods can distinguish these modifications?

The phosphorylation state of ALX4 can significantly affect antibody recognition and biological function interpretation:

• The standard ALX4 Antibody, FITC conjugated targets a specific region (112-216AA) and may not distinguish between phosphorylated and non-phosphorylated forms unless specifically designed to do so.

• To distinguish phosphorylation states:

  • Use phospho-specific ALX4 antibodies in parallel experiments

  • Employ lambda phosphatase treatment on duplicate samples to compare detection before and after dephosphorylation

  • Combine with Phos-tag™ SDS-PAGE to separate phosphorylated from non-phosphorylated forms prior to immunoblotting

• For comprehensive analysis, consider complementary approaches like mass spectrometry to identify specific phosphorylation sites.

• When studying signaling pathways involving ALX4, time-course experiments with pathway activators/inhibitors can help correlate phosphorylation status with functional outcomes.

Phosphorylation can alter protein conformation, potentially masking or revealing epitopes recognized by the antibody. This is especially relevant for transcription factors like ALX4, whose activity is often regulated by post-translational modifications in response to developmental or environmental signals.

What are the common problems encountered when using ALX4 Antibody, FITC conjugated and how can they be resolved?

Researchers commonly encounter several challenges when working with ALX4 Antibody, FITC conjugated:

• Low signal intensity:

  • Increase antibody concentration or incubation time

  • Enhance antigen retrieval methods (for fixed tissues)

  • Ensure proper storage to maintain fluorescence activity

  • Check sample fixation isn't masking epitopes

• High background:

  • Increase blocking time and concentration (10% serum with 1% BSA)

  • Use more extensive washing steps

  • Reduce antibody concentration

  • Ensure samples aren't overexposed during imaging

• Photobleaching:

  • Minimize exposure to light during all steps

  • Use anti-fade mounting media

  • Capture FITC channels first during imaging

  • Consider lower laser power with longer exposure/integration times

• Inconsistent staining:

  • Standardize all protocols including fixation times and temperatures

  • Prepare fresh dilutions of antibody for each experiment

  • Ensure uniform temperature during incubations

  • Process all comparative samples simultaneously

• Non-specific nuclear staining:

  • Perform more stringent blocking with 5% BSA and 0.3M glycine

  • Titrate antibody to find optimal concentration

  • Include additional washing steps with PBS containing 0.1% Tween-20

If problems persist despite these adjustments, validating antibody specificity with knockdown/knockout controls or using alternative antibody clones targeting different epitopes may be necessary .

How can ALX4 Antibody, FITC conjugated be used effectively in flow cytometry applications?

For optimal flow cytometry results with ALX4 Antibody, FITC conjugated:

• Cell preparation:

  • Use single-cell suspensions with viability >90%

  • For intracellular targets like ALX4, proper fixation and permeabilization are critical

  • For nuclear proteins, specialized nuclear permeabilization reagents may yield better results than standard protocols

• Antibody titration:

  • Perform a dilution series (recommended starting range: 1:10-50)

  • Plot signal-to-noise ratio versus antibody concentration to determine optimal dilution

  • Too high concentration can increase non-specific binding; too low results in poor separation

• Controls:

  • Include unstained cells to set baseline fluorescence

  • Use isotype control to identify non-specific binding

  • Include a positive control sample whenever possible

  • Consider fluorescence-minus-one (FMO) controls for multi-color panels

• Data acquisition:

  • Adjust voltage settings so negative populations appear in the first decade of the log scale

  • Collect sufficient events (minimum 10,000, preferably 30,000-50,000) for statistical significance

  • Use appropriate compensation when combining with other fluorophores

• Analysis approach:

  • Gate on intact cells using FSC/SSC, then on single cells using pulse width or height/area plots

  • Use viability dye to exclude dead cells which often show non-specific binding

  • When examining nuclear proteins like ALX4, analyze nuclear size/complexity as a quality control metric

Flow cytometry protocols for transcription factors typically require more rigorous permeabilization than membrane proteins, so specialized nuclear permeabilization buffers are recommended for consistent results .

What is the recommended protocol for immunofluorescence using ALX4 Antibody, FITC conjugated?

A detailed immunofluorescence protocol for ALX4 Antibody, FITC conjugated:

• Cell preparation:

  • Culture cells on coverslips or chamber slides to 70-80% confluence

  • Wash cells twice with pre-warmed PBS

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde in PBS for 15 minutes at room temperature

  • Wash 3 times with PBS, 5 minutes each

  • Permeabilize with 0.25% Triton X-100 in PBS for 10 minutes at room temperature

  • Wash 3 times with PBS, 5 minutes each

• Blocking:

  • Block with 10% normal goat serum and 1% BSA in PBS for 1 hour at room temperature

  • This reduces non-specific binding and improves signal-to-noise ratio

• Primary antibody incubation:

  • Dilute ALX4 Antibody, FITC conjugated 1:500 in PBS containing 10% fetal bovine serum

  • Incubate in a humidified chamber for 1-2 hours at room temperature or overnight at 4°C

  • Protect from light using aluminum foil or dark containers

• Washing:

  • Wash 3 times with PBS, 5 minutes each

  • All washing steps should be performed with gentle agitation

• Nuclear counterstaining:

  • Incubate with DAPI (1 μg/mL in PBS) for 5 minutes at room temperature

  • Wash briefly with PBS

• Mounting:

  • Mount coverslips using anti-fade mounting medium

  • Seal edges with clear nail polish

  • Store slides at 4°C in the dark

• Imaging:

  • Image using appropriate filter sets for FITC (excitation ~495 nm, emission ~519 nm)

  • Capture multiple fields for statistical analysis

  • Include scale bars in all images

For research studying ALX4 in nuclear contexts, z-stack imaging is recommended to fully capture the three-dimensional distribution of this transcription factor within nuclei .

How can ALX4 Antibody, FITC conjugated be integrated into studies of epigenetic regulation?

ALX4 Antibody, FITC conjugated can provide valuable insights into epigenetic regulatory mechanisms:

• Chromatin Immunoprecipitation (ChIP) adaptation:

  • While standard ChIP protocols use unconjugated antibodies, FITC-conjugated antibodies can be utilized in specialized fluorescence-assisted ChIP protocols

  • Use anti-FITC secondary antibodies conjugated to magnetic beads to pull down ALX4-bound chromatin regions

  • Analyze through qPCR or sequencing to identify ALX4 binding sites

• Co-localization with epigenetic marks:

  • Combine ALX4 Antibody, FITC conjugated with antibodies against histone modifications (H3K4me3, H3K27me3, etc.)

  • Use confocal microscopy to assess spatial relationships between ALX4 and specific chromatin states

  • Quantify co-localization using appropriate statistical methods and software

• Analysis during cellular differentiation:

  • Track ALX4 expression and localization during developmental transitions

  • Correlate changes with alterations in chromatin accessibility (ATAC-seq data)

  • Integrate with transcriptomic data to identify ALX4-regulated genes

• Drug studies:

  • Monitor ALX4 distribution following treatment with epigenetic modifiers (HDAC inhibitors, DNA methyltransferase inhibitors)

  • Assess changes in ALX4 binding patterns in response to chromatin remodeling

This antibody is particularly valuable for epigenetics and nuclear signaling research , allowing researchers to visualize dynamic changes in ALX4 localization and interaction with chromatin during development or in response to experimental manipulations.

What are the critical quality control checks when validating a new lot of ALX4 Antibody, FITC conjugated?

When validating a new lot of ALX4 Antibody, FITC conjugated, researchers should perform these essential quality control checks:

• Fluorophore validation:

  • Measure absorbance/emission spectra to confirm FITC conjugation integrity

  • Calculate fluorophore-to-protein ratio to ensure consistent labeling efficiency

  • Compare fluorescence intensity with previous lots using standardized samples

• Specificity testing:

  • Western blot with positive control lysates to confirm correct molecular weight detection

  • Immunofluorescence on known positive and negative cell types

  • Peptide competition assay using recombinant ALX4 protein (112-216AA)

  • Compare staining pattern with unconjugated anti-ALX4 antibody followed by FITC-secondary

• Performance validation:

  • Side-by-side comparison with previous lot on identical samples

  • Titration experiment to confirm optimal working dilution hasn't changed

  • Signal-to-noise ratio assessment across various applications

• Stability testing:

  • Test antibody performance after multiple freeze-thaw cycles

  • Assess stability at working dilution over time (0, 24, 48, 72 hours)

• Documentation:

  • Record lot number, receipt date, validation results

  • Update protocols if new optimal conditions are identified

  • Maintain validation images with acquisition settings for future reference

How does ALX4 Antibody, FITC conjugated compare with other detection methods for studying ALX4?

Researchers have multiple options for studying ALX4, each with distinct advantages and limitations:

When choosing between these approaches, consider:

• The specific research question (protein localization vs. expression level)

• Sample type (fixed tissue vs. live cells)

• Need for quantification precision

• Compatibility with other techniques in your experimental workflow

For studies requiring direct visualization of endogenous ALX4 protein without genetic manipulation, ALX4 Antibody, FITC conjugated offers a good balance of specificity and convenience, particularly for flow cytometry applications where signal-to-noise ratio is a critical parameter .

What are the advanced approaches for studying ALX4 in developmental biology models?

Developmental biology studies of ALX4 require sophisticated approaches to capture spatial and temporal dynamics:

• Lineage tracing techniques:

  • Combine ALX4 Antibody, FITC conjugated with genetic lineage markers

  • Use pulse-chase experiments to track ALX4-expressing cells through development

  • Correlate ALX4 expression with fate determination events

• Organoid models:

  • Apply ALX4 Antibody, FITC conjugated in 3D organoid culture systems

  • Perform live imaging to track ALX4 expression during organoid development

  • Use quantitative image analysis to measure expression gradients

• CRISPR-based approaches:

  • Create ALX4 reporter lines using CRISPR knock-in strategies

  • Generate conditional ALX4 knockout models to study stage-specific functions

  • Use CUT&RUN or CUT&Tag to map ALX4 binding sites genome-wide

• Single-cell analysis:

  • Combine flow cytometry using ALX4 Antibody, FITC conjugated with single-cell RNA-seq

  • Isolate ALX4-positive populations at different developmental stages

  • Create trajectory maps of ALX4-expressing lineages

• Intravital imaging:

  • Adapt ALX4 Antibody, FITC conjugated for in vivo imaging applications

  • Use nanobody-based approaches for improved tissue penetration

  • Implement light-sheet microscopy for reduced phototoxicity during long-term imaging

These advanced approaches enable researchers to move beyond static observations of ALX4 expression toward functional understanding of its role in developmental processes, particularly in neural crest development, skeletal formation, and other contexts where this homeobox transcription factor plays critical regulatory roles .