GBE1 Antibody, FITC conjugated

What is GBE1 and why is it significant in research?

GBE1 (Glucan (1,4-alpha-), Branching Enzyme 1) is an essential enzyme that participates in glycogen biosynthesis alongside glycogenin and glycogen synthase. It functions by generating α-1,6-glucosidic branches from α-1,4-linked glucose chains, thereby increasing the solubility of the glycogen polymer . This branching activity is critical for proper glycogen structure and metabolism.

GBE1 research is significant due to its involvement in several pathological conditions. Mutations in the GBE1 gene lead to glycogen storage disorder type IV (GSDIV), a severe early-onset condition, or adult polyglucosan body disease (APBD), a late-onset neurodegenerative disorder . Recent studies have also implicated GBE1 in cancer progression, particularly in gliomas and lung adenocarcinoma, making it a potential therapeutic target for metabolic cancer therapy .

What are the characteristics of GBE1 antibody, FITC conjugated?

GBE1 antibodies conjugated with FITC typically present the following characteristics:

The FITC conjugation enables visualization through fluorescence microscopy, flow cytometry, and other fluorescence-based detection methods without requiring secondary antibody conjugation steps .

What are the recommended applications for GBE1 antibody, FITC conjugated?

GBE1 antibodies conjugated with FITC can be utilized in multiple research applications, with methodology considerations for each:

• Flow Cytometry (FACS): Optimal for cellular expression analysis at the single-cell level. FITC's excitation/emission properties (499/515 nm) make it compatible with standard 488 nm laser lines . Temperature-dependent uptake studies indicate active transport into cells, as demonstrated in peripheral blood mononuclear cells (PBMCs) .

• Immunofluorescence (IF): Useful for determining subcellular localization of GBE1. Several GBE1 antibodies have been validated for immunofluorescence applications .

• Fluorescence Microscopy: Enables visualization of GBE1 distribution in tissue sections or cell cultures. FITC's bright green fluorescence provides good contrast against DAPI-stained nuclei .

• Cell Sorting: Can be used to isolate GBE1-expressing cells for downstream applications.

The specific dilution requirements vary by application and should be optimized by researchers. For example, some GBE1 antibodies are recommended at 1/10-1/50 dilution for immunofluorescence applications .

How should GBE1 antibody, FITC conjugated be stored and handled?

Proper storage and handling of GBE1 antibody, FITC conjugated is critical for maintaining functionality:

Storage Recommendations:

• Store at -20°C in small aliquots to prevent repeated freeze/thaw cycles

• Protect from light to prevent photobleaching of the FITC fluorophore

• Store in buffer containing stabilizers (typically PBS with glycerol and preservatives)

• Many commercial preparations contain 50% glycerol and 0.03% Proclin-300 as preservative

Handling Guidelines:

• Thaw aliquots completely before use and mix gently (avoid vortexing)

• Maintain antibody on ice or at 4°C during experimental procedures

• Work under reduced light conditions when possible

• Avoid repeated freeze/thaw cycles, which can lead to antibody degradation and loss of FITC signal

• For long-term storage beyond initial aliquoting, ultracold freezers (-80°C) may provide better stability

Researchers should note that FITC is sensitive to pH changes, with optimal fluorescence occurring at slightly alkaline pH (8.0-9.0) .

How can GBE1 antibody, FITC conjugated be utilized to study glycogen storage disorders?

GBE1 antibody, FITC conjugated provides valuable methodological approaches for studying glycogen storage disorders like GSDIV and APBD:

Methodology for Patient Sample Analysis:

• Detection of Mutant Protein Expression: FITC-conjugated GBE1 antibodies can be used to detect and quantify the expression levels of mutant GBE1 protein in patient-derived fibroblasts or peripheral blood mononuclear cells (PBMCs). This approach revealed that certain mutations, such as p.Y329S, result in decreased protein stability and expression .

• Localization Studies: Immunofluorescence using FITC-conjugated GBE1 antibodies can determine if mutations alter the subcellular localization of GBE1, providing insights into disease mechanisms.

• Therapeutic Screening: As demonstrated with the LTKE peptide study, FITC-conjugated peptides can be used alongside GBE1 antibodies to monitor potential therapeutic interventions. The uptake of FITC-labeled peptides was time-dependent and temperature-dependent, suggesting active transport into cells .

• Enzyme Activity Correlation: By combining FITC-labeled GBE1 detection with enzymatic activity assays, researchers can establish direct correlations between protein levels and functional deficits in patient samples. In APBD patient cells, GBE1 activity was enhanced approximately 2-fold when treated with stabilizing peptides .

• Hapten Immunoassay Approach: This technique can be employed to study binding specificity of therapeutic peptides to mutant GBE1, as demonstrated with LTKE-FITC peptide, which showed specific binding to hGBE1-Y329S with an apparent Kd of 18 μM .

These approaches provide mechanistic insights into how GBE1 mutations lead to disease and potential avenues for therapeutic intervention.

What methodological approaches can be used to investigate GBE1's role in cancer using FITC-conjugated antibodies?

Recent research has implicated GBE1 in cancer progression, particularly in gliomas and lung adenocarcinoma. FITC-conjugated GBE1 antibodies can be methodologically applied in several ways to elucidate GBE1's role in cancer:

Cancer Research Methodologies:

• Expression Analysis in Tumor Tissues:

  • Immunohistochemistry and immunofluorescence using GBE1 antibodies on tissue microarrays can correlate GBE1 expression with clinical outcomes

  • Studies have shown that GBE1 expression correlates with poor prognosis in glioma patients

• Metabolic Profiling:

  • GBE1 promotes glioma progression by enhancing aerobic glycolysis through inhibition of fructose-bisphosphatase 1 (FBP1)

  • FITC-conjugated GBE1 antibodies can be used alongside metabolic indicators to track glycolytic changes in cancer cells

• Pathway Analysis:

  • GBE1 has been shown to reduce FBP1 expression through the NF-κB pathway, shifting glucose metabolism patterns in glioma cells to glycolysis and enhancing the Warburg effect

  • Research has demonstrated that hypoxia-induced HIF1α mediates GBE1 upregulation, suppressing FBP1 expression by promoter methylation via NF-κB signaling in lung adenocarcinoma cells

• Immune Regulation Studies:

  • GBE1 knockdown increases expression of chemokines CCL5 and CXCL10 in lung adenocarcinoma cells

  • Supernatants from GBE1 knockdown cells increased recruitment of CD8+ T lymphocytes

  • FITC-conjugated GBE1 antibodies can help track changes in GBE1 expression during immune response modulation

• Flow Cytometry Analysis:

  • Flow cytometry with FITC-conjugated GBE1 antibodies allows quantitative assessment of GBE1 expression changes under different experimental conditions (hypoxia, drug treatment, etc.)

  • This approach can be combined with other markers to establish correlation with cell cycle progression or apoptosis

These methodological approaches provide comprehensive strategies for investigating GBE1's multifaceted roles in cancer progression and potential as a therapeutic target.

What controls and validation procedures should be implemented when using GBE1 antibody, FITC conjugated?

Rigorous validation and appropriate controls are essential for generating reliable data with GBE1 antibody, FITC conjugated:

Validation Procedures:

• Antibody Specificity Validation:

  • Western blot analysis with recombinant GBE1 protein to confirm molecular weight specificity (expected 80-85 kDa band)

  • Testing in multiple cell lines with known GBE1 expression levels

  • Comparison with multiple GBE1 antibodies targeting different epitopes

  • Knockout/knockdown controls where GBE1 expression is reduced using siRNA or CRISPR-Cas9 to confirm signal specificity

• FITC Conjugation Quality Control:

  • Spectral analysis to confirm appropriate excitation/emission profiles (499/515 nm)

  • Degree of labeling (DOL) determination to ensure optimal fluorophore-to-protein ratio

  • Functional binding comparison between conjugated and unconjugated antibody preparations

Essential Experimental Controls:

• Negative Controls:

  • Isotype control antibody conjugated with FITC from the same host species

  • Secondary antibody-only controls when applicable

  • Unstained samples to establish autofluorescence baseline

  • Non-expressing cell lines or tissues

• Positive Controls:

  • Cell lines with confirmed high GBE1 expression (e.g., liver cells, where GBE1 is abundant)

  • Recombinant GBE1-expressing constructs

  • Tissues with known GBE1 expression patterns (liver, muscle)

• Technical Controls:

  • Concentration-matched non-specific IgG-FITC to assess non-specific binding

  • FITC quenching control (photobleaching assessment)

  • Signal calibration using standardized beads (for flow cytometry)

  • Multi-channel compensation when using additional fluorophores

These validation procedures and controls ensure that any observed signals truly represent GBE1 expression and are not artifacts or non-specific binding events.

How can GBE1 antibody, FITC conjugated be used in protein-protein interaction studies?

GBE1 antibody, FITC conjugated can be strategically employed in several methodologies to investigate protein-protein interactions:

Methodological Approaches:

• Co-Immunoprecipitation with Fluorescence Detection:

  • Use GBE1 antibody to pull down protein complexes

  • Analyze interacting partners through standard methods

  • FITC labeling enables direct visualization of complexes without secondary detection

  • This approach has been useful in examining GBE1 interactions with other glycogen metabolism enzymes

• Fluorescence Resonance Energy Transfer (FRET):

  • Pair FITC-conjugated GBE1 antibody with a secondary interacting protein labeled with an appropriate acceptor fluorophore

  • Measure energy transfer as indication of proximity

  • Can detect interactions within 10 nm distance

  • Particularly valuable for detecting dynamic interactions in living cells

• Proximity Ligation Assay (PLA):

  • Use FITC-conjugated GBE1 antibody with secondary antibody against potential interacting protein

  • Rolling circle amplification creates fluorescent spots when proteins are in close proximity

  • This approach has been used to study GBE1 interactions with NF-κB pathway components

• Fluorescence Correlation Spectroscopy (FCS):

  • Analyze movement of FITC-conjugated GBE1 antibody-labeled proteins

  • Changes in diffusion rate can indicate complex formation

  • Provides quantitative binding affinity measurements

• Live Cell Imaging of Protein Complexes:

  • When used with cell-penetrating techniques, FITC-conjugated antibodies can track GBE1 interactions in real time

  • This approach revealed that peptide therapy (LTKE-FITC) binds specifically to mutant GBE1-Y329S in APBD patient cells with an apparent Kd of 18 μM

These methods can help elucidate GBE1's interactions with other proteins in glycogen metabolism pathways and its role in disease mechanisms, particularly how GBE1 influences the NF-κB pathway in cancer progression .

What are the quantitative analysis techniques for GBE1 expression studies using FITC-conjugated antibodies?

Quantitative analysis of GBE1 expression using FITC-conjugated antibodies requires sophisticated methodologies to ensure accurate and reproducible results:

Quantitative Methodologies:

• Flow Cytometry Analysis:

  • Mean Fluorescence Intensity (MFI) measurement provides relative expression levels

  • Standard curve generation using calibration beads enables absolute quantification

  • Multi-parameter analysis correlates GBE1 expression with cell cycle stages or other markers

  • Population gating strategies can identify cell subsets with differential GBE1 expression

  • Essential controls include: unstained cells, isotype controls, and single-color compensation controls

• Fluorescence Microscopy Quantification:

  • Integrated Density Measurement: Total fluorescence within defined cellular regions

  • Signal-to-background ratio calculation for reliable detection above autofluorescence

  • Z-stack acquisition for 3D quantification of total cellular GBE1

  • Colocalization analysis with organelle markers using Pearson's or Mander's coefficients

• High-Content Imaging Analysis:

  • Automated multi-well imaging for high-throughput screening

  • Machine learning algorithms for cell classification based on GBE1 expression patterns

  • Time-lapse imaging to track dynamic changes in GBE1 localization or expression

• Therapeutic Monitoring:

  • Quantitative assessment of GBE1 restoration in patient cells after treatment

  • In APBD patient cells, LTKE peptide treatment resulted in approximately 2-fold increase in mutant enzyme activity compared to untreated cells (from ~5% to >15% of unaffected control)

  • Analysis of dose-response relationships for potential therapeutics

• Western Blot Fluorescence Detection:

  • Direct fluorescence scanning of FITC signal on membranes

  • Linear dynamic range typically spans 2-3 orders of magnitude

  • Standardization against housekeeping proteins or total protein stains

  • Samples from various tissues show GBE1 expression is particularly high in liver and skeletal muscle

These quantitative approaches provide robust methods for analyzing GBE1 expression across different experimental conditions, enabling researchers to make reliable comparisons between normal and disease states or before and after therapeutic interventions.

How can researchers overcome technical challenges when working with GBE1 antibody, FITC conjugated?

Researchers frequently encounter technical challenges when working with FITC-conjugated antibodies for GBE1 detection. Here are methodological solutions to common problems:

Challenge: Photobleaching of FITC Signal

• Solution: Use anti-fade mounting media containing protective agents like n-propyl gallate or p-phenylenediamine

• Methodology: Prepare slides immediately before imaging; minimize exposure time; consider using newer generation dyes with better photostability for critical applications

• Alternative Approach: Consider sequential image acquisition of different fields rather than extended exposure of a single field

Challenge: High Background Fluorescence

• Solution: Optimize blocking conditions and implement additional washing steps

• Methodology: Extend blocking time to 2 hours using 5% BSA or 10% normal serum from the same species as the secondary antibody; use 0.1% Triton X-100 in wash buffers to reduce non-specific membrane binding

• Alternative Approach: Consider autofluorescence quenching agents such as Sudan Black B (0.1-0.3%) or Pontamine Sky Blue (0.5%)

Challenge: Low Signal-to-Noise Ratio

• Solution: Signal amplification techniques

• Methodology: Implement tyramide signal amplification (TSA) which can enhance FITC signal 10-100 fold; alternatively, use biotin-streptavidin systems prior to FITC detection

• Alternative Approach: Consider primary antibody concentration step or longer incubation at 4°C (overnight)

Challenge: Cross-Reactivity Issues

• Solution: Additional validation steps and more stringent controls

• Methodology: Pre-absorb antibody with recombinant proteins of potential cross-reactive targets; use tissues from GBE1 knockout models as negative controls

• Alternative Approach: Compare staining patterns with multiple GBE1 antibodies targeting different epitopes

Challenge: Weak Signal in Fixed Tissues

• Solution: Optimize antigen retrieval methods

• Methodology: Compare heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0); optimize retrieval times

• Alternative Approach: Try proteolytic digestion with enzymes like proteinase K as an alternative retrieval method

Challenge: Variable Results Between Experiments

• Solution: Standardize protocols and implement calibration standards

• Methodology: Use internal reference samples in each experiment; include calibration beads for flow cytometry; normalize to reference genes or proteins

• Alternative Approach: Consider automated systems that minimize operator variability

By implementing these methodological solutions, researchers can optimize their experiments with GBE1 antibody, FITC conjugated to obtain more consistent and reliable results.