C1GALT1C1 Antibody, Biotin conjugated

What is C1GALT1C1 and what is its primary function in cellular biology?

C1GALT1C1, also known as COSMC (Core 1 β3-galactosyltransferase-specific molecular chaperone), functions as a chaperone protein required for the generation of core 1 O-glycan Gal-beta1-3GalNAc-alpha1-Ser/Thr, commonly known as the T antigen. This glycan structure serves as a precursor for many extended O-glycans in glycoproteins . The protein functions as a specific molecular chaperone that assists in the proper folding and stability of core 1 beta-3-galactosyltransferase (C1GALT1), which is essential for initiating complex O-glycan biosynthesis .

C1GALT1C1 is encoded by an X-chromosomal gene, and its proper function is critical for numerous cellular processes. The chaperone activity of C1GALT1C1 ensures that T-synthase (C1GALT1) maintains appropriate enzymatic activity, which is necessary for the production of mature O-glycans required for many cellular functions . Without functional C1GALT1C1, T-synthase activity decreases, leading to a loss of mature O-glycans and potential pathological consequences.

How does C1GALT1C1 differ from C1GALT1, and what is their relationship?

While C1GALT1C1 and C1GALT1 work together in O-glycosylation, they serve distinct functions:

What phenotypes are associated with C1GALT1C1 mutations or deficiency?

Studies in animal models and human cases have revealed several significant phenotypes associated with C1GALT1C1 mutations:

Complete knockout of C1galt1c1 in mice results in embryonic lethality, demonstrating its essential role in development . This lethal phenotype is accompanied by system-wide accumulation of the Tn-antigen (GalNAcα1-O-Ser/Thr/Tyr; CD175), which is the immature precursor glycan to the T-antigen .

Partial loss-of-function models exhibit:

• Thrombocytopenia

• Bleeding diathesis

• Smaller body size

• Severe proteinuric kidney disease with glomerular loss in renal histology

In humans, a germline loss-of-function variant in C1GALT1C1 (c.59C>A, p.Ala20Asp) has been identified in patients with a multisystem disorder characterized by altered protein O-glycosylation . Analysis of X-inactivation patterns in heterozygous female patients showed a skewed pattern, with the wild-type allele preferentially inactivated, resulting in predominant expression of the mutant allele in 78.7% to 89.2% of cells .

What are the optimal conditions for using C1GALT1C1 Antibody, Biotin conjugated in ELISA applications?

For optimal ELISA performance with C1GALT1C1 Antibody, Biotin conjugated, researchers should consider the following methodological approaches:

Buffer Composition:

• Use PBS (0.01M, pH 7.4) as the base buffer to maintain antibody stability

• Add 50% glycerol to prevent freeze-thaw damage and maintain antibody functionality

• Include a preservative such as 0.03% Proclin 300 to prevent microbial contamination

Sample Preparation:

• When detecting recombinant C1GALT1C1, focus on the protein region corresponding to amino acids 27-318, as this represents the immunogenic region used to generate the antibody

• Use freshly prepared samples whenever possible to maintain protein integrity

Blocking Conditions:

• Implement a robust blocking step (typically 1-2 hours at room temperature) using a buffer that minimizes background without interfering with the biotin conjugation

• BSA (1-5%) is often effective for blocking non-specific binding sites

Detection System:

• Utilize streptavidin-coupled detection systems (HRP or fluorescent) that maximize the advantage of the biotin conjugation

• Optimize streptavidin concentration to prevent oversaturation while ensuring adequate signal

Controls:

• Include a purified recombinant C1GALT1C1 protein as a positive control

• Include negative controls (buffer only and unrelated protein samples) to ensure specificity

Since this antibody has been specifically tested and validated for ELISA applications, adhering to these methodological guidelines should produce reliable and reproducible results .

What are the proper storage and handling procedures for maintaining C1GALT1C1 Antibody, Biotin conjugated efficacy?

Proper storage and handling of C1GALT1C1 Antibody, Biotin conjugated is critical for maintaining its immunoreactivity and ensuring experimental reproducibility. Based on manufacturer recommendations, researchers should follow these guidelines:

Short-term Storage:

• Maintain at 2-8°C (refrigerated) for up to 2 weeks

• Avoid repeated freeze-thaw cycles, which can degrade the antibody and reduce its efficacy

Long-term Storage:

• Store at -20°C or -80°C for extended periods

• Upon receipt of the antibody, immediately divide into small working aliquots before freezing to minimize freeze-thaw cycles

Thawing Protocol:

• Thaw frozen aliquots on ice or in a refrigerator (4°C)

• Once thawed, use immediately for best results or keep at 4°C if using within 1-2 days

Working Solution Handling:

• Keep the antibody on ice during experiment setup

• Avoid vortexing vigorously, which can cause protein denaturation

• Gently mix by inversion or mild pipetting

Transportation:

• If transferring between laboratories, maintain cold chain conditions using dry ice or cold packs

• Monitor temperature during transport to ensure antibody viability

Protection from Light:

• As this is a biotin-conjugated antibody, minimize exposure to light during handling to prevent potential photobleaching of the biotin conjugate

Buffer Considerations:

• The antibody is supplied in PBS with 50% glycerol and 0.03% Proclin 300

• Avoid introducing substances that might interfere with antibody activity or the biotin conjugate

Following these storage and handling protocols will help ensure optimal antibody performance and experimental consistency over time.

How can researchers validate the specificity of C1GALT1C1 Antibody, Biotin conjugated?

Validating antibody specificity is crucial for ensuring experimental rigor. For C1GALT1C1 Antibody, Biotin conjugated, researchers should employ a multi-faceted approach:

Positive Control Validation:

• Use purified recombinant human C1GALT1C1 protein (specifically amino acids 27-318) as a positive control in ELISA

• Compare signals between samples known to express C1GALT1C1 and those that do not

Competitive Blocking:

• Pre-incubate the antibody with excess purified C1GALT1C1 antigen before application to samples

• A significant reduction in signal indicates specific antibody-antigen interaction

Western Blot Analysis (if adapting for WB):

• Run side-by-side samples of wild-type cells and those with C1GALT1C1 knockdown/knockout

• Observe band presence/absence at the expected molecular weight (~36 kDa for human C1GALT1C1)

Immunoprecipitation Followed by Mass Spectrometry:

• Use the antibody to immunoprecipitate C1GALT1C1 from cell lysates

• Confirm the identity of pulled-down proteins by mass spectrometry

Cross-reactivity Assessment:

• Test the antibody against closely related proteins, particularly C1GALT1, to ensure it does not cross-react

• This is especially important given the functional relationship between C1GALT1C1 and C1GALT1

Knockout/Knockdown Validation:

• Compare antibody signals in wild-type samples versus those with CRISPR/siRNA-mediated reduction of C1GALT1C1

• A specific antibody will show reduced or absent signal in knockout/knockdown samples

Validation in Multiple Systems:

• Test the antibody across different experimental platforms beyond ELISA

• Confirm reactivity with human samples as specified in the product information

How can C1GALT1C1 Antibody, Biotin conjugated be used to study the relationship between O-glycosylation patterns and disease phenotypes?

C1GALT1C1 Antibody, Biotin conjugated offers several sophisticated approaches to investigate O-glycosylation's role in disease pathogenesis:

Tissue Microarray Analysis:

• Apply the antibody to tissue microarrays containing samples from patients with suspected O-glycosylation disorders

• Compare C1GALT1C1 expression patterns with known disease biomarkers

• Quantify correlation between C1GALT1C1 levels and disease severity parameters

Glycophenotyping:

• Combine C1GALT1C1 Antibody detection with lectin profiling to simultaneously assess:
  a) Chaperone abundance (via C1GALT1C1 antibody)
  b) O-glycan structure alterations (via specific lectins)

• This dual approach can reveal whether disease phenotypes correlate with chaperone availability, glycan structural changes, or both

Flow Cytometry Applications:

• Develop flow cytometry protocols using the biotin-conjugated antibody to quantify C1GALT1C1 levels in patient-derived cells

• Sort cell populations based on C1GALT1C1 expression levels to study functional consequences

Cell-Specific O-glycosylation Analysis:

• In tissues showing germline C1GALT1C1 mutations (like the c.59C>A variant), use the antibody to:
  a) Determine which cell types express mutant C1GALT1C1
  b) Correlate with Tn-antigen accumulation (using anti-Tn antibodies)
  c) Link to cell-specific pathology

Correlation with X-inactivation Patterns:

• In female patients with C1GALT1C1 mutations, use the antibody to analyze the relationship between:
  a) X-inactivation skewing (determined genetically)
  b) C1GALT1C1 protein expression
  c) Disease severity

• This approach can help explain clinical heterogeneity in X-linked conditions

This methodological framework enables researchers to establish mechanistic connections between aberrant O-glycosylation and disease manifestations, potentially identifying new therapeutic targets for conditions like the multisystem disorder described in patients with C1GALT1C1 mutations .

What methodologies can be employed to investigate the interaction between C1GALT1C1 and C1GALT1 using antibody-based approaches?

Investigating the critical chaperone-enzyme interaction between C1GALT1C1 and C1GALT1 requires sophisticated methodological approaches:

Co-immunoprecipitation (Co-IP) Studies:

• Use the C1GALT1C1 Antibody, Biotin conjugated to pull down C1GALT1C1 complexes

• Detect co-precipitated C1GALT1 using separate anti-C1GALT1 antibodies

• Analyze binding efficiency under different conditions (pH, temperature, ion concentration)

• Compare wild-type versus mutant C1GALT1C1 (e.g., the p.Ala20Asp variant) binding to C1GALT1

Proximity Ligation Assay (PLA):

• Combine C1GALT1C1 Antibody, Biotin conjugated with anti-C1GALT1 antibody

• Use streptavidin-linked and secondary antibody-linked DNA oligonucleotides

• Visualize protein proximity (<40 nm) through rolling circle amplification

• Quantify interaction events in different cellular compartments

FRET/BRET Analysis:

• Develop Förster/Bioluminescence Resonance Energy Transfer systems using:
  a) C1GALT1C1 Antibody, Biotin conjugated (with streptavidin-fluorophore)
  b) Fluorescently labeled anti-C1GALT1 antibody

• Measure energy transfer efficiency as indication of protein proximity

• Compare interaction dynamics in normal versus disease states

Surface Plasmon Resonance (SPR):

• Immobilize C1GALT1C1 Antibody, Biotin conjugated on streptavidin-coated SPR chips

• Use antibody to capture C1GALT1C1 in defined orientation

• Flow purified C1GALT1 over the surface

• Determine binding kinetics (kon, koff) and affinity (KD)

• Compare wild-type and mutant C1GALT1C1 binding parameters

Functional Reconstitution Assays:

• Immunodeplete C1GALT1C1 from cellular extracts using the antibody

• Assess remaining T-synthase (C1GALT1) activity

• Restore activity by adding back purified C1GALT1C1

• Quantify chaperone efficiency through restoration of enzyme activity

These methodological approaches can elucidate the molecular details of the C1GALT1C1-C1GALT1 interaction, providing insights into how mutations affect chaperone function and subsequent O-glycosylation patterns in both normal physiology and disease states.

How can researchers utilize C1GALT1C1 Antibody, Biotin conjugated to study X-inactivation patterns in heterozygous females?

The X-chromosomal location of C1GALT1C1 makes it particularly valuable for studying X-inactivation patterns, especially in heterozygous females carrying mutations:

Dual Immunofluorescence Protocol:

• Use C1GALT1C1 Antibody, Biotin conjugated to detect total C1GALT1C1 protein

• Simultaneously employ mutation-specific antibodies (if available) or FISH probes targeting the mutant allele

• Develop a quantitative scoring system for cells expressing:
  a) Wild-type protein only
  b) Mutant protein only
  c) Both proteins (if complete X-inactivation has not occurred)

Single-Cell Analysis Workflow:

• Perform flow cytometry using the biotin-conjugated antibody to isolate cells based on C1GALT1C1 expression levels

• Sort cells into high and low C1GALT1C1-expressing populations

• Analyze genomic DNA from sorted populations to determine which X chromosome is active in each fraction

• Correlate with functional O-glycosylation analysis (e.g., Tn-antigen levels)

Tissue-Specific X-Inactivation Assessment:

• Apply the antibody to tissue sections from female patients with heterozygous C1GALT1C1 mutations

• Quantify cellular mosaicism across different tissues

• Compare observed protein expression patterns with genetic X-inactivation analysis

• This approach can explain tissue-specific phenotypes in X-linked disorders

Correlation with Disease Severity:

• In female patients with skewed X-inactivation patterns (as observed in the case with 78.7-89.2% cells expressing the mutant allele)

• Use the antibody to quantify the actual percentage of cells expressing functional C1GALT1C1 protein

• Correlate protein expression levels with clinical parameters

• Develop predictive models linking X-inactivation ratios to disease outcomes

Longitudinal X-Inactivation Studies:

• Track changes in C1GALT1C1 expression patterns over time using the antibody

• Investigate age-related skewing in X-inactivation patterns

• Correlate with disease progression or amelioration

These methodological approaches provide researchers with powerful tools to understand how X-chromosome inactivation influences C1GALT1C1 expression and subsequent O-glycosylation patterns, particularly in heterozygous females with C1GALT1C1 mutations who show variable disease expression.

What are common sources of background signal when using C1GALT1C1 Antibody, Biotin conjugated, and how can they be mitigated?

When working with C1GALT1C1 Antibody, Biotin conjugated, researchers may encounter several sources of background signal that can be systematically addressed:

Protocol Optimization:

• Perform antibody titration experiments to determine the minimum concentration that provides specific signal

• Implement a step-wise troubleshooting approach, changing only one variable at a time

• Include controls that allow discrimination between various sources of background:

  • Omit primary antibody

  • Omit streptavidin detection reagent

  • Include known negative samples

This systematic approach to troubleshooting will help researchers optimize signal-to-noise ratios when using C1GALT1C1 Antibody, Biotin conjugated, ensuring reliable and reproducible experimental results.

How can researchers optimize C1GALT1C1 Antibody, Biotin conjugated for applications beyond ELISA?

While the C1GALT1C1 Antibody, Biotin conjugated has been validated for ELISA applications , researchers may adapt it for other techniques with appropriate optimization:

Immunohistochemistry (IHC) Adaptation:

• Antigen Retrieval Optimization:

  • Test multiple retrieval methods (heat-induced with citrate buffer pH 6.0, EDTA buffer pH 9.0, or enzymatic retrieval)

  • Optimize retrieval duration (10-30 minutes)

• Blocking Considerations:

  • Block endogenous biotin using commercial biotin blocking kits before antibody application

  • Include avidin/biotin blocking steps for tissues with high endogenous biotin (liver, kidney)

• Detection System Selection:

  • Use streptavidin-HRP systems with amplification for low-abundance targets

  • Consider tyramide signal amplification for maximum sensitivity

• Counterstaining Adaptation:

  • Adjust counterstain intensity to provide contrast without obscuring specific signals

Flow Cytometry Protocol Development:

• Cell Preparation:

  • Optimize fixation method (2-4% paraformaldehyde) and duration (10-20 minutes)

  • Test permeabilization reagents (0.1-0.5% saponin, 0.1-0.3% Triton X-100) for intracellular staining

• Staining Protocol:

  • Determine optimal antibody concentration through titration experiments

  • Include dead cell discrimination dyes to eliminate false positives

  • Implement compensation controls when used in multicolor panels

• Signal Amplification:

  • Consider secondary streptavidin-fluorophore conjugates with varying brightness

  • Test photostability of different fluorophores for prolonged analysis

Immunoprecipitation Adaptation:

• Bead Selection:

  • Use streptavidin-coated magnetic beads for efficient capture

  • Determine optimal bead-to-sample ratio through titration experiments

• Pre-clearing Strategy:

  • Implement sample pre-clearing with unconjugated beads to reduce non-specific binding

  • Include appropriate isotype controls

• Elution Conditions:

  • Develop gentle elution methods to maintain protein-protein interactions

  • Consider competitive elution with biotin for native complex isolation

These methodological adaptations should always include appropriate validation steps to confirm that the antibody maintains specificity for C1GALT1C1 in each new application, as performance characteristics may vary across different techniques.

How should researchers interpret variations in C1GALT1C1 expression across different tissue and cell types?

When analyzing C1GALT1C1 expression patterns detected with the biotin-conjugated antibody, researchers should consider several interpretive frameworks:

Physiological Variation Analysis:

• Compare C1GALT1C1 expression levels with known O-glycosylation requirements in different tissues

• Tissues with extensive mucin production (gastrointestinal tract, respiratory epithelium) may show higher baseline expression

• Correlate expression with C1GALT1 levels to assess the chaperone-to-enzyme ratio across tissues

X-Inactivation Considerations:

• In female subjects, mosaic expression patterns may reflect X-inactivation patterns

• Quantify cell-to-cell variability within tissues to assess X-inactivation mosaicism

• Compare with known X-inactivation patterns in the analyzed tissues

Developmental Context:

• Interpret expression changes in the context of developmental stage

• Consider that knockout studies in mice show embryonic lethality , suggesting critical developmental roles

• Analyze temporal expression patterns during organogenesis and tissue maturation

Pathological Correlation Framework:

• When examining diseased tissues, correlate C1GALT1C1 expression with:

  • Tn-antigen accumulation (indicator of incomplete O-glycosylation)

  • Disease severity markers

  • Specific pathological features (e.g., kidney glomerular changes, thrombocytopenia)

Statistical Analysis Approaches:

• Employ quantitative image analysis for immunohistochemistry data

• Use appropriate statistical tests based on data distribution:

  • Parametric tests (t-test, ANOVA) for normally distributed data

  • Non-parametric alternatives (Mann-Whitney, Kruskal-Wallis) for non-normal distributions

• Control for multiple comparisons when analyzing numerous tissue types

Data Visualization:

• Develop tissue expression maps showing relative C1GALT1C1 abundance

• Use heatmaps to visualize expression patterns across multiple tissues and conditions

• Create correlation matrices linking expression with functional O-glycosylation markers

What approaches can researchers use to analyze the relationship between C1GALT1C1 expression and O-glycosylation profiles?

Establishing connections between C1GALT1C1 expression and O-glycosylation patterns requires integrated analytical approaches:

Multi-Modal Analytical Framework:

• Combine C1GALT1C1 protein detection (using the biotin-conjugated antibody) with:

  • Lectin binding assays (specific for O-glycan structures)

  • Mass spectrometry glycomic profiling

  • T-synthase activity assays

• This comprehensive approach allows correlation between chaperone presence and functional outcomes

Correlation Analysis Methodology:

• Calculate Pearson or Spearman correlation coefficients between:

  • C1GALT1C1 expression levels

  • T-synthase (C1GALT1) activity

  • T-antigen abundance

  • Tn-antigen accumulation

• Identify threshold effects where changes in C1GALT1C1 levels lead to nonlinear changes in glycosylation

Perturbation Analysis:

• Manipulate C1GALT1C1 expression experimentally (knockdown, overexpression)

• Quantify resulting changes in:

  • T-synthase stability (protein half-life)

  • Enzyme activity (glycosyltransferase assays)

  • O-glycan profiles (mass spectrometry)

• Establish dose-response relationships between chaperone levels and functional outcomes

Glycoprotein-Specific Assessment:

• Analyze specific glycoprotein targets known to be O-glycosylated

• Compare glycoform distributions in contexts of varying C1GALT1C1 expression

• Identify particularly sensitive glycoprotein substrates

Pathway Integration Analysis:

• Place C1GALT1C1 expression data within broader cellular contexts:

  • ER stress response

  • Protein quality control machinery

  • Glycosylation enzyme expression networks

• Develop models explaining how C1GALT1C1 integrates with other chaperones and glycosylation machinery

Clinical Correlation Framework:

• In patient samples with identified C1GALT1C1 mutations (such as the c.59C>A variant) :

  • Quantify residual C1GALT1C1 activity

  • Measure T-synthase stability and activity

  • Profile O-glycan structures

  • Correlate with clinical phenotypes

This analytical framework enables researchers to establish mechanistic connections between C1GALT1C1 expression and functional O-glycosylation outcomes, providing insights into both normal physiology and disease pathogenesis.