Customize GAUT1 Antibody

Description

Introduction to GAUT1 Antibody

The GAUT1 antibody is a critical research tool for studying the function and interactions of GAUT1 (galacturonosyltransferase 1), a key enzyme in synthesizing homogalacturonan (HG), the primary pectic polysaccharide in plant cell walls. HG is essential for cell wall structure, development, and environmental responses. The antibody facilitates biochemical, immunological, and subcellular localization studies of GAUT1, enabling insights into its role in plant cell wall biosynthesis and protein complex assembly .

Target Protein and Specificity

GAUT1: A type II transmembrane glycosyltransferase (GT family 8) with an α-1,4-galacturonosyltransferase activity. It synthesizes HG by transferring galacturonic acid (GalA) from UDP-GalA to oligogalacturonides .
Post-Translational Modification: GAUT1 undergoes N-terminal cleavage (removing its transmembrane domain), resulting in a mature protein of ~60 kDa (detected via Western blot) .
Complex Formation: GAUT1 forms a disulfide-linked heterodimer with GAUT7, a non-catalytic homolog, which anchors GAUT1 in the Golgi .

Antibody Production and Reactivity

Source: Polyclonal antisera generated against recombinant GAUT1 fragments .
Reactivity: Recognizes both native (immunoabsorption) and denatured (Western blot) GAUT1, confirming its specificity .

Immunodepletion and Activity Assays

Experiment	Purpose	Key Findings	Source
Immunoabsorption	Deplete GAUT1 from Arabidopsis membrane fractions to confirm its role in HG synthesis.	GAUT1 antiserum removed ~98.9% of GalAT activity; residual activity confirmed via EPG treatment (which cleaves HG).
Western Blotting	Detect GAUT1 in solubilized membrane fractions.	Identified a ~60 kDa band corresponding to processed GAUT1; absent in immunodepleted fractions.

Protein Interaction Studies

Method	Purpose	Key Findings	Source
Co-IP (Co-Immunoprecipitation)	Validate GAUT1-GAUT7 interaction.	GAUT1 and GAUT7 co-immunoprecipitate; confirmed via anti-GAUT1 and anti-GAUT7 antibodies.
Bimolecular Fluorescence Complementation (BiFC)	Visualize GAUT1-GAUT7 interaction in vivo.	Fluorescence signal observed in Golgi membranes, confirming complex formation.

Core Components

GAUT1:GAUT7 Complex: The catalytic core of HG:GalAT activity. Proteomics analyses (LC-MS/MS) confirmed GAUT1 and GAUT7 as the only stable components under non-reducing SDS-PAGE conditions .
Putative Interactors: Twelve additional proteins co-immunoprecipitated with the GAUT1:GAUT7 complex, suggesting broader functional associations (e.g., glycosyltransferases, Golgi-localized proteins) .

Functional Insights

Catalytic Role: The GAUT1:GAUT7 complex selectively elongates oligogalacturonides (OGAs) but not other pectic precursors (e.g., RG-I, RG-II) .
Golgi Anchoring: GAUT7 retains processed GAUT1 in the Golgi, as shown by transient expression of GAUT1-GFP in Nicotiana benthamiana .

Table 1: GAUT1 Antibody Applications in Key Experiments

Application	Method	Outcome
Activity Confirmation	Immunoabsorption with anti-GAUT1	Depletion of HG:GalAT activity; residual activity confirmed via EPG digestion.
Interaction Validation	Co-IP with anti-GAUT1/anti-GAUT7	GAUT1 and GAUT7 co-purify; disulfide bonds stabilize the complex.
Localization Studies	Western blotting	Detection of ~60 kDa GAUT1 in Golgi-enriched fractions.

Table 2: Proteomics Analysis of GAUT1:GAUT7 Complex

Component	Role	Evidence
GAUT1	Catalytic subunit (α-1,4-GalAT)	Immunodepletion abolishes HG synthesis; cleaved N-terminal domain.
GAUT7	Membrane anchor	Co-IP and BiFC confirm interaction; no GalAT activity when expressed alone.
Putative Interactors	Uncharacterized (12 proteins)	Identified via LC-MS/MS; potential roles in pectin synthesis or trafficking.

Product Specs

Buffer

**Preservative:** 0.03% Proclin 300
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4

Form

Liquid

Lead Time

Made-to-order (14-16 weeks)

Synonyms

GAUT1 antibody; JS36 antibody; LGT1 antibody; At3g61130 antibody; T20K12.30Polygalacturonate 4-alpha-galacturonosyltransferase antibody; EC 2.4.1.43 antibody; Alpha-1,4-galacturonosyltransferase 1 antibody; Galacturonosyltransferase 1 antibody; Like glycosyl transferase 1 antibody

Target Names

GAUT1

Uniprot No.

Q9LE59

Target Background

Function

GAUT1 Antibody is involved in pectin biosynthesis. It catalyzes the transfer of galacturonic acid from uridine 5'-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan.

Gene References Into Functions

Galacturonosyltransferases (GAUT1) is a homogalacturonan galacturonosyltransferase belonging to the GAUT1-related gene family, playing a crucial role in pectin biosynthesis. [GAUT1] PMID: 16540543

Database Links

KEGG: ath:AT3G61130

STRING: 3702.AT3G61130.1

UniGene: At.969

Protein Families

Glycosyltransferase 8 family

Subcellular Location

Golgi apparatus membrane; Single-pass type II membrane protein.

Tissue Specificity

Expressed in seedlings, inflorescences, flowers, siliques, pollen, roots, stems and leaves.

Q&A

Here’s a structured collection of FAQs tailored for researchers studying GAUT1 antibodies in academic contexts, synthesized from peer-reviewed sources and methodological insights:

Advanced Research Questions

How to resolve discrepancies in GAUT1 molecular weight observations?

Condition	Observed MW (kDa)	Predicted MW (kDa)	Explanation	Source
Reducing SDS-PAGE	60	77.4	Post-translational cleavage of N-terminal transmembrane domain
Non-reducing SDS-PAGE	185	-	Disulfide-linked GAUT1:GAUT7 heterocomplex

What strategies address contradictory data on GAUT1’s catalytic role?

Analytical framework:
- Conduct ChiP-Seq alongside transcriptomics to distinguish direct transcriptional targets from indirect regulatory effects .
- Compare mutant lines (e.g., gaut1 knockout) with wild-type plants to isolate GAUT1-specific contributions to HG biosynthesis .
- Use structural modeling to assess GAUT1’s active site (e.g., conserved DxD motifs in galacturonosyltransferases) .

How to differentiate GAUT1’s enzymatic activity from its structural role in the GAUT1:GAUT7 complex?

Experimental design:
- Perform in vitro GalAT assays using purified GAUT1 vs. GAUT1:GAUT7 complexes .
- Apply subcellular fractionation to isolate Golgi membranes and quantify HG synthesis rates .
- Analyze cleavage mutants (e.g., GAUT1ΔN167) to determine if catalytic activity requires N-terminal processing .

Methodological Troubleshooting

Why might GAUT1 antibody fail to detect protein in transient expression systems?

Critical checks:
- Confirm GAUT7 co-expression for proper GAUT1 stabilization and Golgi retention .
- Optimize detergent conditions for membrane protein extraction (e.g., 1% Triton X-100).
- Validate antibody reactivity against recombinant truncated GAUT1 (e.g., residues 168–633) .

How to confirm GAUT1’s role in nitrogen-responsive gene networks?

Integrated workflow:
- Apply TARGET (Transient Assay Reporting Genome-wide Effects) to identify GAUT1-regulated genes under nitrogen perturbation .
- Cross-reference RNA-seq data with DNA-binding assays (e.g., DAP-Seq) to map GAUT1 promoter interactions .

Data Interpretation Guidelines

How to assess GAUT1’s evolutionary conservation across plant species?

Comparative analysis:
- Align GAUT1 orthologs using PhyloNet to identify conserved domains (e.g., C-terminal catalytic region) .
- Test functional complementation in heterologous systems (e.g., gaut1 mutants rescued with Populus GAUT1) .

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GAUT1 Antibody