At4g31140 Antibody

What is the At4g31140 gene and why develop antibodies against it?

At4g31140 is a gene in Arabidopsis thaliana that encodes a glycosyl hydrolase family 17 protein . Researchers develop antibodies against this protein to study its expression, localization, and function in plant cells. The protein appears to be involved in plant responses to environmental stressors, particularly salinity stress, making it an important target for research in plant stress physiology . Antibodies against At4g31140 allow researchers to perform various immunological techniques including western blotting, immunoprecipitation, and immunofluorescence microscopy to determine protein abundance, interactions, and subcellular localization.

What are the main experimental applications for At4g31140 antibodies?

At4g31140 antibodies are primarily used in the following experimental applications:

• Western blot analysis to detect and quantify At4g31140 protein expression levels

• Immunoprecipitation (IP) to isolate the protein and study its interaction partners

• Chromatin immunoprecipitation (ChIP) if the protein has DNA-binding properties

• Immunofluorescence microscopy to determine subcellular localization

• ELISA assays for quantitative detection

Similar to other research antibodies used in plant studies, protocols typically need to be optimized for specificity in Arabidopsis tissues, with appropriate controls to validate antibody specificity .

How should I validate the specificity of At4g31140 antibodies?

Validating the specificity of At4g31140 antibodies is crucial for reliable research outcomes. A comprehensive validation approach should include:

• Western blot analysis comparing wild-type plants with At4g31140 knockout mutants (similar to the approach used for SKB1 antibody validation)

• Testing antibody reactivity against recombinant At4g31140 protein

• Performing peptide competition assays

• Conducting immunoprecipitation followed by mass spectrometry

• Testing cross-reactivity with related glycosyl hydrolase family proteins

A properly validated antibody should show:

• A single band of expected molecular weight in wild-type samples

• Absence of this band in knockout mutants

• No significant cross-reactivity with other proteins

What are the optimal protein extraction protocols for At4g31140 detection in plant tissues?

For optimal detection of At4g31140 in plant tissues, consider the following extraction protocol:

• Harvest 20-day-old seedlings grown under controlled conditions (similar to protocols used for histone protein extraction)

• Grind tissue in liquid nitrogen to a fine powder

• Extract proteins using a buffer containing:

  • 50 mM Tris-HCl, pH 7.5

  • 150 mM NaCl

  • 1% Triton X-100 or 0.1% SDS

  • 1 mM EDTA

  • Protease inhibitor cocktail

• Centrifuge at 12,000 × g for 15 minutes at 4°C

• Collect supernatant and quantify protein concentration

For membrane-associated proteins, consider using a modified extraction protocol with detergents appropriate for membrane protein solubilization, similar to methods used for tonoplast proteins .

How should I optimize western blot conditions for At4g31140 antibody detection?

Optimizing western blot conditions for At4g31140 antibody detection requires careful consideration of several parameters:

• Sample preparation:

  • Use 20-30 μg of total protein extract

  • Include reducing agent (DTT or β-mercaptoethanol) in sample buffer

  • Heat samples at 95°C for 5 minutes before loading

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels

  • Transfer to PVDF membrane at 100V for 60-90 minutes

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat dry milk in TBST for 1 hour

  • Dilute primary antibody 1:1000 to 1:2000 in blocking solution

  • Incubate overnight at 4°C

  • Wash 3×10 minutes with TBST

  • Incubate with 1:10,000 to 1:20,000 dilution of peroxidase-conjugated secondary antibody

  • Wash 3×10 minutes with TBST

• Detection:

  • Use enhanced chemiluminescence (ECL) detection system

  • Optimize exposure time based on signal intensity

What controls should be included when using At4g31140 antibodies?

When using At4g31140 antibodies, include the following controls to ensure experimental validity:

• Positive control:

  • Recombinant At4g31140 protein or extract from tissues known to express the protein

  • Samples from plants overexpressing the target protein

• Negative control:

  • Protein extracts from At4g31140 knockout mutants (similar to skb1 mutants used as negative controls)

  • Pre-immune serum in place of primary antibody

• Loading control:

  • Antibodies against housekeeping proteins (e.g., actin, tubulin)

  • Ponceau S staining of the membrane

• Specificity control:

  • Peptide competition assay

  • "No primary antibody" control

How can At4g31140 antibodies be used in ChIP assays to study protein-DNA interactions?

While At4g31140 is identified as a glycosyl hydrolase, if research indicates DNA-binding properties, ChIP assays could be performed following protocols similar to those used for histone modification studies :

• Cross-linking:

  • Harvest 20-day-old seedlings

  • Cross-link protein-DNA complexes with 1% formaldehyde for 10 minutes

  • Quench with 0.125 M glycine

• Chromatin preparation:

  • Extract nuclei and isolate chromatin

  • Sonicate to generate DNA fragments of 200-500 bp

• Immunoprecipitation:

  • Incubate sonicated chromatin with At4g31140 antibody (typically 2-5 μg)

  • Include appropriate controls (e.g., IgG, input DNA)

  • Capture antibody-protein-DNA complexes using protein A/G beads

• Washing and elution:

  • Perform stringent washes to remove non-specific binding

  • Elute and reverse cross-links

• DNA purification and analysis:

  • Purify DNA and analyze by qPCR, sequencing, or other methods

This approach would allow identification of genomic regions potentially bound by At4g31140, similar to how SKB1 association with the FLC promoter was demonstrated .

What approaches can be used to study At4g31140 protein interactions using antibodies?

To study At4g31140 protein interactions, the following approaches using antibodies can be employed:

• Co-immunoprecipitation (Co-IP):

  • Use At4g31140 antibodies to pull down the protein along with its interacting partners

  • Identify partners by mass spectrometry

  • Verify specific interactions by reciprocal Co-IP

• Proximity labeling coupled with immunoprecipitation:

  • Express At4g31140 fused to a proximity labeling enzyme (BioID or TurboID)

  • After biotin labeling of proximal proteins, use antibodies to confirm specific interactions

• Yeast two-hybrid validation:

  • Use antibodies to confirm interactions identified in Y2H screens

  • Perform Co-IP to validate interactions in planta

• Bimolecular Fluorescence Complementation (BiFC) validation:

  • Use antibodies to confirm expression of fusion proteins in BiFC assays

How can At4g31140 antibodies be used to study protein post-translational modifications?

To study post-translational modifications (PTMs) of At4g31140, researchers can employ several antibody-based approaches:

• Immunoprecipitation followed by mass spectrometry:

  • Use At4g31140 antibodies to immunoprecipitate the protein

  • Analyze the purified protein by mass spectrometry to identify PTMs

  • Compare PTM profiles under different conditions or treatments

• Modification-specific antibodies:

  • If specific PTMs (phosphorylation, glycosylation, etc.) are identified, develop or obtain antibodies specific to these modified forms

  • Use these alongside general At4g31140 antibodies to track changes in modification status

• 2D gel electrophoresis combined with western blotting:

  • Separate proteins based on both isoelectric point and molecular weight

  • Use At4g31140 antibodies to detect different protein forms representing various PTMs

The search results suggest At4g31140 may potentially have N-glycosylation sites, as it's listed in the context of plant proteins . This could be particularly relevant as the search results indicate it's a glycosyl hydrolase family protein, which might itself be subject to glycosylation .

How can I address non-specific binding issues with At4g31140 antibodies?

Non-specific binding is a common challenge when working with antibodies. To address this issue with At4g31140 antibodies:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, non-fat dry milk, commercial blockers)

  • Increase blocking time to 2 hours or more

  • Add 0.1-0.5% Tween-20 to reduce non-specific hydrophobic interactions

• Adjust antibody dilution:

  • Test a range of primary antibody dilutions (1:500 to 1:5000)

  • Reduce incubation time or temperature

• Modify washing protocol:

  • Increase number and duration of washes

  • Use higher salt concentration in wash buffers (up to 500 mM NaCl)

  • Add 0.1% SDS to wash buffer for highly specific washes

• Pre-absorb the antibody:

  • Incubate antibody with extract from knockout plants

  • Use commercially available pre-absorption kits

• Affinity purification:

  • Purify antibody against immobilized antigen to increase specificity

For immunofluorescence applications, include appropriate controls using pre-immune serum and secondary antibody-only samples to determine background signal levels .

What strategies can overcome weak or absent signals when using At4g31140 antibodies?

When facing weak or absent signals with At4g31140 antibodies, consider implementing these strategies:

• Sample preparation optimization:

  • Test different protein extraction methods

  • Use protease inhibitor cocktails to prevent degradation

  • Enrich for the cellular fraction where At4g31140 is expected to localize

• Signal enhancement techniques:

  • Use high-sensitivity detection systems (e.g., enhanced chemiluminescence plus)

  • Consider tyramide signal amplification for immunohistochemistry

  • Use signal enhancer solutions before primary antibody incubation

• Antigen retrieval methods:

  • For fixed tissues, test heat-induced or enzymatic antigen retrieval

  • For membrane proteins, optimize detergent concentrations in extraction buffers

• Antibody concentration adjustments:

  • Increase primary antibody concentration

  • Extend primary antibody incubation time (overnight at 4°C)

  • Test different lots or sources of antibody

• Sample loading modifications:

  • Increase amount of total protein loaded

  • Consider using tissue or conditions where At4g31140 expression is upregulated, possibly in response to salinity stress

How can At4g31140 antibodies be applied in cell type-specific transcriptional response studies?

Antibodies against At4g31140 can be valuable tools in studying cell type-specific transcriptional responses, particularly in the context of stress responses in plants :

• Immunohistochemistry for spatial expression analysis:

  • Use fluorescently-labeled secondary antibodies to visualize At4g31140 distribution across different cell types

  • Compare expression patterns under normal versus stress conditions

  • Combine with cell type-specific markers to correlate expression with specific tissues

• Cell sorting followed by western blotting:

  • Use fluorescence-activated cell sorting (FACS) to isolate specific cell populations

  • Perform western blotting with At4g31140 antibodies to quantify protein levels in different cell types

• Laser capture microdissection combined with immunoassays:

  • Isolate specific cell types using laser capture

  • Extract proteins and perform dot blots or micro-western arrays

  • Quantify At4g31140 protein levels across different cell types

• Chromatin immunoprecipitation in specific cell types:

  • If At4g31140 has DNA-binding properties, perform cell type-specific ChIP

  • Use techniques similar to those described for SKB1 ChIP assays , but with cell type-specific isolation

This approach could help identify differential roles of At4g31140 across various plant tissues and cell types, particularly in response to environmental stressors like salinity .

How can machine learning approaches improve At4g31140 antibody-based research?

Recent advances in machine learning offer opportunities to enhance antibody-based research for targets like At4g31140:

• Prediction of antibody-antigen binding:

  • Machine learning models can predict which epitopes of At4g31140 are most likely to generate specific antibodies

  • These predictions can guide epitope selection for antibody development

• Image analysis automation:

  • Machine learning algorithms can analyze immunofluorescence images to quantify protein localization patterns

  • This enables high-throughput analysis of At4g31140 expression across multiple conditions

• Active learning for experimental design:

  • Similar to the approach described for antibody-antigen binding prediction , active learning can be used to optimize experimental designs

  • This approach could reduce the number of required experiments by up to 35%

• Prediction of post-translational modifications:

  • Machine learning can predict likely sites of PTMs on At4g31140

  • These predictions can be validated using antibody-based approaches

How can At4g31140 antibody studies be combined with genetic approaches?

Integrating At4g31140 antibody studies with genetic approaches creates powerful research strategies:

• Verification of knockout and knockdown lines:

  • Use At4g31140 antibodies to confirm protein absence in T-DNA insertion mutants

  • Similar to verification of SKB1 knockout in skb1-1 and skb1-2 lines

  • Quantify protein reduction in RNAi or CRISPR lines

• Complementation studies:

  • Use antibodies to confirm protein expression in complementation lines

  • Correlate protein levels with phenotype rescue

• Protein localization in genetic backgrounds:

  • Examine At4g31140 localization in various mutant backgrounds

  • Identify genetic components affecting protein trafficking or stability

• Protein-protein interaction network mapping:

  • Combine Co-IP using At4g31140 antibodies with genetic interaction studies

  • Identify both physical and genetic interaction partners

A comprehensive approach might include generating T-DNA insertion lines in At4g31140 (similar to the skb1-1 and skb1-2 lines described ), then using RT-PCR to confirm disruption of gene expression and western blotting with At4g31140 antibodies to confirm absence of protein.

What are the best approaches for studying At4g31140 in relation to plant stress responses?

Given the context of At4g31140 appearing in research related to plant salinity responses , the following approaches are recommended:

• Expression analysis under stress conditions:

  • Use At4g31140 antibodies for western blot analysis of protein levels under various stresses

  • Compare protein levels across a time course of stress exposure

  • Correlate protein abundance with stress severity

• Subcellular relocalization studies:

  • Use immunofluorescence to track potential changes in At4g31140 localization during stress

  • Compare with known stress response proteins

• Protein modification analysis:

  • Examine changes in post-translational modifications of At4g31140 during stress

  • Immunoprecipitate the protein and analyze by mass spectrometry

• Protein-protein interaction changes:

  • Use Co-IP to identify stress-specific interaction partners

  • Compare interactome under normal versus stress conditions

These approaches would help elucidate the role of At4g31140 in plant stress responses, building upon the transcriptional data indicating its involvement in salinity responses .