At2g43240 Antibody

What is At2g43240 and why is it significant for plant research?

At2g43240 is a gene in Arabidopsis thaliana that encodes the CMP-sialic acid transporter 2 (also known as CMP-SA-Tr 2 or CMP-Sia-Tr 2). This protein consists of 406 amino acids and is involved in the transport of CMP-sialic acid, which is crucial for various cellular processes . Understanding this transporter is significant for plant glycobiology research, particularly in studies examining cell membrane transport mechanisms and glycosylation pathways in plants.

What is known about the protein structure of At2g43240?

The At2g43240 protein consists of 406 amino acids with a specific sequence as documented in the protein databases (NP_001118506.1 and NP_181853.3) . The protein contains multiple transmembrane domains typical of membrane transporters. As a CMP-sialic acid transporter, it likely adopts a tertiary structure with multiple membrane-spanning regions to facilitate the transport of its substrate across cellular membranes.

How does At2g43240 expression vary across plant tissues?

While the provided search results don't specifically detail the expression pattern, researchers typically approach this question through techniques like quantitative immunohistochemistry across different plant tissues. Similar to approaches used in the Human Protein Atlas for human proteins, expression patterns can be determined by using validated antibodies against At2g43240 in various plant tissue samples . This provides spatial information on protein distribution, which complements quantitative data obtained through other proteomic methods.

What types of At2g43240 antibodies are available for research?

Currently, there are mouse monoclonal antibodies available targeting different regions of the At2g43240 protein. These include:

• X-Q8GY97-N: A combination of mouse monoclonal antibodies targeting the N-terminus

• X-Q8GY97-C: A combination of mouse monoclonal antibodies targeting the C-terminus

• X-Q8GY97-M: A combination of mouse monoclonal antibodies targeting the middle (non-terminus) sequence

Each antibody combination is derived from three synthetic peptides representing the respective protein region.

How should researchers validate At2g43240 antibodies before experimental use?

Antibody validation should follow multiple strategies as recommended by the International Working Group for Antibody Validation (IWGAV). For At2g43240 antibodies, researchers should consider:

• Orthogonal validation: Compare protein expression using an antibody-independent method (e.g., mass spectrometry)

• Independent antibody validation: Compare results using two different antibodies targeting non-overlapping regions of At2g43240 (e.g., using both N-terminus and C-terminus targeting antibodies)

• Genetic validation: Test antibody specificity in knockout/knockdown models, if available

• Expression validation: Verify antibody performance in samples with known expression levels

• Pre-absorption validation: Assess specificity by pre-absorbing the antibody with the immunizing peptide

A multi-strategy approach provides greater confidence in antibody specificity and reliability.

What criteria determine whether an At2g43240 antibody has been sufficiently validated?

An At2g43240 antibody should be considered sufficiently validated when:

• It demonstrates specific binding to the target protein in multiple applications

• Results are reproducible across different experimental setups

• It shows minimal cross-reactivity with other proteins

• The antibody performs consistently in the specific application (e.g., Western blot, immunohistochemistry)

• At least two independent validation strategies have yielded consistent results

What is the recommended protocol for using At2g43240 antibodies in Western blot analysis?

While specific optimized protocols for At2g43240 antibodies are not provided in the search results, the general approach should include:

• Sample preparation: Extract proteins from plant tissue using appropriate buffers maintaining protein integrity

• Protein separation: Run proteins on SDS-PAGE (10-12% gel recommended for a 406 AA protein)

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane

• Blocking: Block with 5% non-fat milk or BSA in TBST

• Primary antibody: Incubate with At2g43240 antibody at optimized dilution (the ELISA titer of 10,000 suggests a starting dilution of 1:1000-1:5000)

• Secondary antibody: Incubate with anti-mouse HRP-conjugated secondary antibody

• Detection: Visualize using chemiluminescence

• Validation: Confirm specificity through appropriate controls

How can At2g43240 antibodies be optimized for immunohistochemistry in plant tissues?

For immunohistochemistry applications:

• Tissue fixation: Fix plant tissues in paraformaldehyde or other suitable fixatives

• Tissue processing and sectioning: Process tissues and prepare thin sections (4-10 μm)

• Antigen retrieval: Perform heat-induced or enzymatic antigen retrieval if necessary

• Blocking: Block endogenous peroxidases and non-specific binding sites

• Primary antibody: Apply At2g43240 antibody at optimized dilution (typically start with 1:100-1:500)

• Secondary antibody and detection: Use appropriate detection system (e.g., HRP-DAB)

• Counterstaining: Apply appropriate counterstain for tissue context

• Controls: Include negative controls (no primary antibody) and positive controls if available

Optimization should follow similar processes to those used in the Human Protein Atlas, with antibody dilution and antigen retrieval conditions adjusted specifically for plant tissues .

What applications besides Western blot and IHC are suitable for At2g43240 antibodies?

Based on the characteristics of the available antibodies, other potential applications include:

• Immunoprecipitation: For isolation of At2g43240 protein complexes

• Immunofluorescence: For subcellular localization studies

• Flow cytometry: For analysis of protein expression in protoplasts

• ELISA: For quantitative measurement of protein levels

• ChIP (if relevant): For studying protein-DNA interactions if At2g43240 has any DNA-binding capabilities

Each application requires specific optimization and validation approaches.

What controls are essential when using At2g43240 antibodies?

Essential controls include:

• Negative controls:

  • No primary antibody control

  • Isotype control (irrelevant antibody of the same isotype)

  • Pre-immune serum control

• Positive controls:

  • Recombinant At2g43240 protein

  • Tissues/cells known to express the protein

  • Overexpression system

• Specificity controls:

  • Peptide competition/blocking studies

  • Knockout/knockdown validation if available

  • Multiple antibodies targeting different epitopes

How can researchers address cross-reactivity concerns with At2g43240 antibodies?

To address potential cross-reactivity:

• Sequence alignment analysis: Identify proteins with sequence similarity to At2g43240

• Multiple antibody approach: Compare results using antibodies targeting different epitopes of At2g43240

• Mass spectrometry verification: Confirm antibody specificity through immunoprecipitation followed by mass spectrometry

• Peptide blocking: Perform competitive blocking with the immunizing peptide

• Enhanced validation: Utilize orthogonal methods to verify specificity

The specificity of available At2g43240 antibodies has been addressed during production by selecting antigen sequences with the lowest possible identity to proteins from other genes, followed by affinity purification and validation against multiple protein epitopes .

What are common pitfalls when interpreting At2g43240 antibody results?

Common pitfalls include:

• Overinterpreting weak signals without proper controls

• Failure to distinguish between specific and non-specific binding

• Incorrect attribution of signals from related protein family members

• Not accounting for post-translational modifications affecting epitope recognition

• Misinterpreting subcellular localization due to fixation artifacts

• Overlooking the impact of sample preparation on protein detection

• Not validating results with orthogonal methods

How can At2g43240 antibodies be integrated with other omics approaches?

Researchers can integrate antibody-based detection of At2g43240 with:

• Transcriptomics: Correlate protein expression with mRNA levels

• Proteomics: Combine with mass spectrometry for comprehensive protein network analysis

• Metabolomics: Link At2g43240 expression with metabolite profiles, particularly sialic acid-containing compounds

• Interactomics: Use antibodies for co-immunoprecipitation to identify interaction partners

• Phenomics: Correlate protein expression with phenotypic traits

This multi-omics approach provides a more comprehensive understanding of the protein's role in plant biology .

What methodological approaches can resolve contradictory results from different At2g43240 antibodies?

When facing contradictory results:

• Epitope mapping: Determine the exact binding sites of different antibodies

• Cross-validation: Use orthogonal, antibody-independent methods

• Independent antibody validation: Compare results using antibodies targeting different regions (N-terminus, C-terminus, and middle region)

• Genetic validation: Test in knockout/knockdown systems

• Expression systems: Validate in heterologous expression systems

• Protocol optimization: Adjust experimental conditions for each antibody

• Statistical analysis: Implement robust statistical methods to evaluate reproducibility

How can researchers quantitatively assess At2g43240 protein expression?

For quantitative analysis:

• Western blot densitometry: Measure relative protein levels with proper loading controls

• Quantitative immunohistochemistry: Analyze staining intensity and cell type distribution

• ELISA: Develop quantitative assays using the available monoclonal antibodies

• Mass spectrometry: Use targeted proteomics with antibody enrichment

• Flow cytometry: Quantify protein levels in individual cells or protoplasts

Each method requires appropriate calibration and controls for accurate quantification.