GBX2 Antibody, FITC conjugated

What is GBX2 and what is its biological significance?

GBX2 (Gastrulation and brain-specific homeobox protein 2) is a homeobox protein that functions as a transcription factor involved in cell pluripotency and differentiation during embryonic development . It plays an essential role in normal development of the midbrain and anterior hindbrain regions . During neural development, GBX2 helps establish the mid/hindbrain boundary (MHB) by forming an expression boundary with Otx2 and negatively regulating Otx2 expression along the anterior-posterior axis . GBX2 is primarily expressed in the anterior hindbrain during development and in the adult brain, spleen, and female genital tract . Research indicates it likely regulates genes involved in establishing early anterior/posterior patterning in the neural plate, making it a crucial molecule for studying neurogenesis and brain development .

What are the key specifications of GBX2 Antibody, FITC conjugated?

The GBX2 Antibody, FITC conjugated is a polyclonal antibody raised in rabbits against recombinant human GBX2 protein (specifically amino acids 65-248) . The antibody has the following specifications:

How does FITC conjugation affect antibody functionality?

FITC (Fluorescein Isothiocyanate) conjugation allows direct visualization of GBX2 protein in immunofluorescence techniques without requiring a secondary antibody . The FITC fluorophore has excitation/emission maxima wavelengths of approximately 493 nm/522 nm, resulting in green fluorescence . While conjugation provides advantages for direct detection, researchers should be aware that the conjugation process may slightly affect binding affinity or specificity compared to unconjugated antibodies. Additionally, FITC is somewhat susceptible to photobleaching compared to other fluorophores, so samples should be protected from excessive light exposure during storage and imaging procedures. For quantitative applications, appropriate controls should be included to account for potential differences in antibody performance due to conjugation.

What are the optimal dilutions and conditions for using GBX2 Antibody, FITC conjugated in immunofluorescence experiments?

• Fix cells with 4% paraformaldehyde (as demonstrated with SK-N-SH cells in some protocols)

• Permeabilize with 0.1-0.5% Triton X-100 in PBS

• Block with 1-5% BSA or normal serum from the same species as the secondary antibody

• Incubate with diluted GBX2-FITC antibody (start with 1:100 dilution)

• Wash thoroughly with PBS (3-5 times)

• Counterstain nuclei with DAPI if desired

• Mount with anti-fade mounting medium

• Store slides at 4°C protected from light to prevent photobleaching

The antibody has been successfully tested on HeLa cells , and data from various neural cell lines including Neuro-2a and SK-N-SH suggests good performance in neuronal models .

Can this GBX2 antibody be used for Western blotting, and what protocol modifications are needed compared to the unconjugated version?

While the FITC-conjugated GBX2 antibody is primarily optimized for immunofluorescence applications, the unconjugated version of this antibody has been validated for Western blotting . If using the FITC-conjugated version for Western blotting, several modifications to standard protocols are necessary:

• Use a specialized imaging system capable of detecting fluorescence directly from membranes

• Protect membranes from light during all incubation and washing steps

• Consider using a higher concentration (approximately 2-3× higher than unconjugated antibody)

• Expected band size is 37 kDa (predicted) with observed bands between 33-37 kDa

In Western blot applications using the unconjugated variant at 1/1000 dilution, bands were successfully detected in various mouse cell lines including Neuro-2a, NIH/3T3, RAW 264.7, and C2C12 at 30 μg of whole cell extract . When using 10% SDS-PAGE gels with ECL detection systems, clear bands at the expected molecular weight were observed .

How should samples be prepared for optimal GBX2 detection in neural tissues?

For optimal detection of GBX2 in neural tissues, the following preparation steps are recommended:

• Tissue fixation: Use 4% paraformaldehyde in PBS for 24-48 hours, followed by paraffin embedding or cryoprotection in 30% sucrose for frozen sections

• Section thickness: 5-10 μm for paraffin sections; 10-20 μm for frozen sections

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) for 15-20 minutes is recommended for paraffin sections

• Permeabilization: 0.2-0.3% Triton X-100 in PBS for 10-15 minutes

• Blocking: 5% normal goat serum with 1% BSA in PBS for 1-2 hours at room temperature

• Primary antibody: Dilute GBX2-FITC antibody 1:50-1:100 in blocking solution and incubate overnight at 4°C

• Wash steps: Extensive washing (4-5 times, 5 minutes each) with PBS-T (0.1% Tween-20 in PBS)

Since GBX2 is primarily expressed in specific regions of the developing brain, with particular emphasis on the anterior hindbrain region, special attention should be paid to proper anatomical orientation and identification of these regions during tissue processing and analysis .

What are the most common issues encountered when using GBX2 Antibody, FITC conjugated, and how can they be resolved?

Several common issues may arise when using this antibody:

• Weak or no signal:

  • Increase antibody concentration (use lower dilution)

  • Extend incubation time to overnight at 4°C

  • Ensure proper antigen retrieval for fixed tissues

  • Verify target expression in your samples (GBX2 is tissue-specific)

  • Check fluorescence microscope settings for FITC detection (excitation ~493 nm, emission ~522 nm)

• High background or non-specific staining:

  • Increase blocking time or blocking agent concentration

  • Use more stringent washing (longer and more wash steps)

  • Decrease antibody concentration (use higher dilution)

  • Include 0.1-0.3% Triton X-100 in antibody diluent to reduce non-specific binding

  • Ensure samples are protected from light to prevent autofluorescence

• Photobleaching:

  • Minimize exposure to light during all steps

  • Use anti-fade mounting medium containing DABCO or similar compounds

  • Capture images quickly and minimize exposure during microscopy

  • Consider using lower intensity excitation or shorter exposure times

• Cross-reactivity:

  • Include appropriate controls (secondary antibody only, isotype control)

  • Validate specificity using GBX2 knockout or knockdown samples

What controls should be included when using GBX2 Antibody, FITC conjugated in experimental designs?

A robust experimental design should include the following controls:

• Positive control: Tissues or cells known to express GBX2, such as:

  • Neuronal cell lines (Neuro-2a, SK-N-SH)

  • Developing brain tissue, particularly from regions of the anterior hindbrain

  • HeLa cells (which have been validated for this antibody)

• Negative control: Tissues or cells known not to express GBX2, or:

  • GBX2 knockout or knockdown samples where available

  • Irrelevant tissues (e.g., adult liver which does not express significant GBX2)

• Technical controls:

  • Omission of primary antibody to assess autofluorescence

  • Isotype control (FITC-conjugated rabbit IgG) at the same concentration

  • Blocking peptide competition assay to demonstrate specificity

  • Concentration gradient to determine optimal antibody dilution

Proper controls are essential for accurate interpretation of results, particularly in developmental studies where GBX2 expression shows distinct spatial and temporal patterns .

How can researchers validate the specificity of this GBX2 antibody in their experimental system?

Validation of antibody specificity is crucial for reliable research outcomes. Researchers can employ these methods:

• Genetic approach:

  • Use GBX2 knockout/knockdown models and confirm loss of signal

  • Overexpress GBX2 and confirm increased signal intensity

  • Use siRNA-mediated knockdown to show reduced signal correlating with reduced GBX2 expression

• Molecular approach:

  • Western blot analysis to confirm single band at the expected molecular weight (33-37 kDa)

  • RNA expression correlation - compare immunostaining patterns with in situ hybridization results

  • Co-immunoprecipitation followed by mass spectrometry to confirm antibody is pulling down GBX2 protein

• Technical validation:

  • Compare staining pattern with other validated anti-GBX2 antibodies

  • Peptide competition assay using the immunizing peptide (amino acids 65-248 of human GBX2)

  • Cross-validate with multiple detection methods (IF, WB, IHC)

How can GBX2 Antibody, FITC conjugated be utilized in studies of neural development and the mid/hindbrain boundary?

The GBX2 Antibody, FITC conjugated provides valuable opportunities for studying neural development, particularly the establishment and maintenance of the mid/hindbrain boundary (MHB), through several advanced approaches:

• Dual immunofluorescence studies:

  • Co-staining with Otx2 antibodies to visualize the precise GBX2/Otx2 boundary formation in the developing neural tube

  • Combined with markers of isthmic organizer activity (Fgf8, Wnt1) to study molecular interactions at the MHB

  • Time-course studies to trace dynamic changes in GBX2 expression domains during critical developmental windows

• Lineage tracing experiments:

  • Using the GBX2-FITC antibody in combination with genetic fate mapping to correlate GBX2 expression with cellular identity and migratory patterns

  • Analysis of clonal relationships between GBX2-expressing progenitors and their differentiated progeny

• Perturbation studies:

  • Examining GBX2 expression after genetic or pharmacological manipulation of signaling pathways involved in MHB formation

  • Studying compensatory mechanisms in conditional knockout models with temporal resolution

• Quantitative analysis:

  • Measuring the precise spatial relationships between GBX2 and other developmental markers along the neural tube

  • Computational modeling of GBX2/Otx2 boundary formation using quantitative imaging data

Since GBX2 negatively regulates Otx2 expression along the anterior-posterior axis , these studies can provide insights into the molecular mechanisms underlying brain regionalization and the establishment of neuronal identities.

What are the considerations for using this antibody in flow cytometry applications for neural progenitor cell analysis?

While the primary validated applications for this GBX2-FITC antibody are ELISA and immunofluorescence , researchers interested in adapting it for flow cytometry should consider:

• Cell preparation:

  • Optimize fixation and permeabilization conditions (4% PFA followed by 0.1-0.3% Triton X-100 or commercial permeabilization buffers)

  • Single-cell suspensions must maintain cellular integrity while allowing antibody access to nuclear GBX2

  • Consider gentler detachment methods (e.g., Accutase instead of trypsin) for sensitive neural progenitors

• Antibody titration:

  • Perform detailed titration experiments (starting range: 1:20 to 1:200)

  • Include appropriate compensation controls for spectral overlap with other fluorophores

  • Signal-to-noise ratio optimization is critical for nuclear transcription factors like GBX2

• Gating strategy:

  • Use forward/side scatter properties to identify intact cells

  • Include nuclear staining (e.g., DRAQ5) to confirm nuclear localization

  • Consider co-staining with neural progenitor markers (Nestin, Sox2) for population identification

• Controls specific to flow cytometry:

  • Fluorescence-minus-one (FMO) controls

  • Isotype control at matching concentration

  • Positive control samples with known GBX2 expression levels

  • Negative control samples lacking GBX2 expression

• Technical considerations:

  • FITC can be susceptible to photobleaching, so minimize light exposure during processing

  • Consider fixation-induced autofluorescence in the FITC channel

  • Standardize voltage settings using calibration beads

How can this antibody be used in research investigating the role of GBX2 in pathological conditions?

GBX2 has important developmental functions and may be implicated in pathological conditions. The GBX2 Antibody, FITC conjugated can be utilized in several research contexts:

• Neurodevelopmental disorders:

  • Investigating GBX2 expression patterns in animal models of neurodevelopmental disorders

  • Comparing GBX2 expression in patient-derived induced pluripotent stem cells (iPSCs) differentiated into neural lineages

  • Assessing GBX2/Otx2 boundary formation in developmental disorder models

• Cancer research:

  • Analyzing GBX2 expression in neuronal and non-neuronal tumors

  • Studying the relationship between GBX2 expression and cancer stem cell properties

  • Investigating GBX2 as a potential biomarker for specific tumor subtypes or stages

• Regenerative medicine:

  • Monitoring GBX2 expression during directed differentiation of stem cells into specific neuronal subtypes

  • Assessing the role of GBX2 in neural repair processes after injury

  • Studying GBX2 in cellular reprogramming experiments

• High-resolution imaging techniques:

  • Super-resolution microscopy to visualize subcellular localization of GBX2

  • Live-cell imaging using membrane-permeable FITC-conjugated antibody fragments to track dynamic changes

  • Spatial transcriptomics combined with GBX2 immunofluorescence to correlate protein expression with transcriptional profiles

Each application requires careful optimization of antibody concentration, sample preparation protocols, and appropriate controls to ensure reliable and reproducible results.

Can the GBX2 Antibody, FITC conjugated be used effectively in multiplex immunofluorescence studies?

This GBX2 Antibody, FITC conjugated can be incorporated into multiplex immunofluorescence studies with several important considerations:

• Spectral compatibility:

  • FITC has excitation/emission maxima at 493nm/522nm , yielding green fluorescence

  • Ideal companion fluorophores include red (e.g., Cy3, Alexa Fluor 594) and far-red (e.g., Cy5, Alexa Fluor 647) to minimize spectral overlap

  • Avoid fluorophores with significant spectral overlap like PE or BODIPY

• Staining protocol optimization:

  • Sequential staining may be necessary if antibodies are from the same host species

  • Consider using Zenon labeling technology or direct conjugates for multiple rabbit antibodies

  • Validate each antibody individually before combining in multiplex panels

• Imaging considerations:

  • Use narrow bandpass filters to minimize bleed-through

  • Perform single-color controls for spectral unmixing

  • Consider linear unmixing algorithms for closely overlapping fluorophores

• Recommended multiplex combinations:

  • GBX2-FITC with Otx2 (red fluorophore) to visualize boundary formation

  • GBX2-FITC with neural markers like NeuN, GFAP (far-red fluorophores)

  • GBX2-FITC with proliferation markers (Ki67) for developmental studies

What approaches can be used to quantify GBX2 expression levels in experimental models?

Quantitative analysis of GBX2 expression using this antibody can be performed through several methodological approaches:

• Image-based quantification:

  • Measure fluorescence intensity in defined regions of interest (ROIs)

  • Quantify nuclear vs. cytoplasmic localization ratios

  • Perform cell counting of GBX2-positive vs. negative populations

  • Use automated image analysis software (ImageJ, CellProfiler) with standardized macros

• Flow cytometry-based quantification:

  • Measure mean fluorescence intensity (MFI) of GBX2-FITC signal

  • Quantify the percentage of GBX2-positive cells in different experimental conditions

  • Use calibration beads to standardize fluorescence measurements across experiments

• Protein-level quantification:

  • Western blot analysis using the unconjugated version of the antibody for comparison

  • Correlate fluorescence intensity with protein levels determined by other methods

• Standardization approaches:

  • Include standard curve samples with known GBX2 expression levels

  • Use reference housekeeping proteins or universal standardization methods

  • Employ quantitative fluorescence standards in each experiment

• Technical considerations for accurate quantification:

  • Control for cell size, density, and morphology differences between samples

  • Account for potential photobleaching during image acquisition

  • Ensure linear range of detection is not exceeded

  • Use biological and technical replicates for statistical validity

How can researchers integrate ChIP-seq data with GBX2 immunofluorescence to understand transcriptional networks?

Integrating ChIP-seq data with GBX2 immunofluorescence provides powerful insights into GBX2's transcriptional regulatory networks. While this FITC-conjugated antibody is optimized for immunofluorescence, researchers can use an integrated approach:

• Sequential experimental design:

  • Perform ChIP-seq using a ChIP-validated GBX2 antibody to identify genome-wide binding sites

  • Follow with GBX2-FITC immunofluorescence to visualize protein expression patterns in the same experimental model

  • Correlate binding data with expression patterns across developmental timepoints or experimental conditions

• Multi-omics integration approaches:

  • Overlay GBX2 ChIP-seq peaks with ATAC-seq data to identify accessible chromatin regions

  • Correlate GBX2 binding sites with RNA-seq expression data of potential target genes

  • Use GBX2-FITC immunofluorescence to validate expression of identified target genes through co-localization studies

• Transcriptional network analysis:

  • Identify enriched transcription factor motifs co-occurring with GBX2 binding sites

  • Validate protein-protein interactions through co-immunoprecipitation or proximity ligation assays

  • Use GBX2-FITC in combination with antibodies against predicted cofactors for co-localization studies

• Functional validation:

  • Overexpress or knock down GBX2 and assess changes in chromatin accessibility and target gene expression

  • Use CRISPR-based approaches to modify GBX2 binding sites and monitor effects on target gene expression

  • Correlate changes in GBX2 binding with alterations in cellular phenotypes through immunofluorescence

This integrated approach allows researchers to connect GBX2's genome-wide binding patterns with its spatial and temporal expression patterns, providing comprehensive understanding of its role in transcriptional regulation during development and in disease states.