At5g19175 Antibody

What is the most effective validation approach for At5g19175 antibody specificity?

Antibody validation is a critical first step in any experimental design. For plant antibodies, the gold standard validation approach involves testing in both wild-type and knockout/mutant lines. Similar to validation performed for other plant antibodies, researchers should:

• Perform Western blot analysis with protein extracts from wild-type and mutant/knockout plants

• Include peptide competition assays to confirm epitope specificity

• Test reactivity against recombinant target protein

• Verify target size matches predicted molecular weight

• Consider cross-reactivity with closely related proteins

Protein sequencing and immunoblotting can provide definitive confirmation of antibody specificity, as demonstrated in studies with other plant antibodies where target antigens were identified through these methods .

What protein extraction methods are optimal for At5g19175 detection?

The extraction method significantly impacts antibody detection efficiency. Based on methodological approaches used for other plant proteins:

For membrane-associated proteins, consider microsomal fractionation through differential centrifugation, which has proven effective for isolating both membrane-bound and soluble protein fractions in Arabidopsis . This approach would be particularly relevant if At5g19175 has membrane association, as plant proteins often partition between multiple cellular compartments.

What are the recommended immunoblotting conditions for At5g19175 antibody?

Optimal immunoblotting conditions must be empirically determined, but established protocols for plant antibodies provide a starting point:

• Protein loading: 3-20 μg total protein per lane (start with 3 μg for abundant proteins)

• Initial antibody dilution: 1:1000 to 1:5000 in TBS-T with 5% BSA or non-fat milk

• Incubation: Overnight at 4°C with gentle agitation

• Washing: 4-6 times with TBS-T (0.1% Tween-20)

• Secondary antibody: Anti-rabbit HRP at 1:5000-1:10,000 dilution

• Detection: Enhanced chemiluminescence (ECL)

For plant proteins, membrane selection (PVDF vs. nitrocellulose) can significantly impact detection sensitivity. PVDF membranes typically provide better retention of hydrophobic proteins .

How should sample preparation be optimized for different plant tissues?

Different plant tissues require specific extraction approaches to maximize protein yield and maintain antigen integrity:

Tissue-specific optimization is critical, as demonstrated in immunodetection studies of plant proteins where extraction conditions significantly impacted antibody binding efficiency .

How can At5g19175 antibody be stored to maintain long-term activity?

Proper storage is essential for antibody longevity:

• Long-term storage: Aliquot and store at -80°C to prevent freeze-thaw cycles

• Working solution: Store at 4°C for up to 2 weeks with 0.02% sodium azide

• Stabilizers: Consider adding BSA (1 mg/ml) to prevent adsorption to surfaces

• Avoid contamination: Use sterile techniques when handling

• Monitor activity: Periodically test antibody performance with positive controls

Documentation of storage conditions and freeze-thaw cycles should be maintained to track potential performance degradation over time.

How can At5g19175 antibody be used to study protein-protein interactions?

For investigating protein interactions, multiple complementary approaches can be employed:

• Co-immunoprecipitation (Co-IP):

  • Lyse tissues in non-denaturing buffer with 0.5-1% NP-40 or Triton X-100

  • Pre-clear lysate with Protein A/G beads

  • Incubate with At5g19175 antibody (2-5 μg per mg of total protein)

  • Capture complexes with Protein A/G beads

  • Analyze precipitated proteins by mass spectrometry or immunoblotting

• In vivo interaction validation:

  • Perform Bimolecular Fluorescence Complementation (BiFC) in protoplasts

  • Use At5g19175 antibody to confirm expression levels

  • Visualize interactions at subcellular resolution

Studies with other plant proteins have successfully utilized these approaches to document protein interactions, such as the MAPKKK5-PBL27 complex formation at the plasma membrane in Arabidopsis protoplasts .

What methods can resolve contradictory results when using At5g19175 antibody?

When facing inconsistent results, systematic troubleshooting is essential:

• Antibody validation assessment:

  • Sequence the immunoprecipitated protein to confirm identity

  • Test multiple antibody lots if available

  • Validate with alternative detection methods

• Experimental variables analysis:

  • Document protein extraction efficiency across methods

  • Evaluate post-translational modifications affecting epitope recognition

  • Consider tissue-specific expression patterns

  • Assess protein stability under different conditions

• Biological context evaluation:

  • Determine if protein levels change with growth conditions or developmental stage

  • Test if pathogen exposure or immune signaling affects protein abundance

  • Examine if protein localization changes under different conditions

Conflicting results often stem from biological variables rather than technical issues, as seen in studies where protein interactions were condition-dependent .

How can At5g19175 antibody be used to study subcellular localization?

For precise localization studies, consider:

• Subcellular fractionation:

  • Separate microsomal and soluble fractions through differential centrifugation

  • Further fractionate microsomes on density gradients

  • Analyze fractions by immunoblotting with At5g19175 antibody

  • Include organelle-specific markers for validation

• Immunofluorescence microscopy:

  • Fix tissues with 4% paraformaldehyde

  • Permeabilize with 0.1-0.5% Triton X-100

  • Block with 3-5% BSA or normal serum

  • Incubate with At5g19175 antibody (1:100-1:500)

  • Detect with fluorophore-conjugated secondary antibody

  • Co-stain with organelle markers

This combined approach was effectively used to demonstrate the dual localization of MAPKKK5 to both plasma membrane and cytosolic compartments in plant cells .

How does plant immunity activation affect At5g19175 protein dynamics?

To investigate protein dynamics during immune responses:

• Treatment protocol:

  • Treat seedlings with immune elicitors (e.g., 10 μM chitin oligomers)

  • Collect tissues at multiple time points (0, 5, 15, 30, 60 minutes)

  • Extract proteins under native conditions

  • Analyze by immunoblotting with At5g19175 antibody

• Protein complex dynamics:

  • Perform co-immunoprecipitation before and after elicitor treatment

  • Assess changes in interaction partners

  • Evaluate post-translational modifications (phosphorylation, ubiquitination)

• Localization changes:

  • Monitor protein redistribution following immune activation

  • Document association/dissociation with membrane compartments

Studies with plant immune components have revealed dynamic changes in protein complexes after elicitor treatment, with some interactions being disrupted specifically after chitin but not flagellin detection .

What is the relationship between gene expression and protein abundance for At5g19175?

To correlate transcript and protein levels:

• Parallel analysis protocol:

  • Extract RNA and protein from the same tissue samples

  • Perform RT-qPCR for transcript quantification

  • Use At5g19175 antibody for protein quantification by immunoblotting

  • Normalize protein levels to appropriate housekeeping controls

• Discrepancy investigation:

  • Assess protein stability through cycloheximide chase experiments

  • Investigate post-transcriptional regulation mechanisms

  • Consider proteasomal degradation (test with MG132 treatment)

Studies have shown that protein levels of some plant components are regulated through proteasome-dependent mechanisms, with proteasome inhibitor MG132 treatment resulting in increased protein accumulation .

What immunoprecipitation protocol works best for At5g19175 protein complexes?

For effective immunoprecipitation:

For plant proteins, gentle lysis conditions are critical for maintaining complex integrity, as demonstrated in studies of immune signaling complexes .

How can At5g19175 antibody be used for immunohistochemistry in plant tissues?

For immunohistochemical detection:

• Tissue preparation:

  • Fix tissue in 4% paraformaldehyde for 2-4 hours

  • Dehydrate and embed in paraffin or resin

  • Section at 5-10 μm thickness

• Antigen retrieval:

  • Citrate buffer (pH 6.0) heat treatment

  • Enzymatic digestion with proteinase K (1-10 μg/ml)

• Immunostaining:

  • Block with 5% normal serum and 0.3% Triton X-100

  • Primary antibody: 1:100-1:500 dilution, overnight at 4°C

  • Secondary antibody: 1:200-1:500, 1-2 hours at room temperature

  • Counterstain with DAPI for nuclear visualization

• Controls:

  • Omit primary antibody (negative control)

  • Use tissue from knockout plants (specificity control)

This approach allows visualization of protein distribution across different cell types, similar to the differential staining observed with other antibodies in distinct tissue regions .

What are the best approaches for quantitative analysis of At5g19175 protein levels?

For quantitative protein analysis:

• Quantitative immunoblotting:

  • Include a standard curve of recombinant protein

  • Load equal amounts of total protein (validate with total protein stain)

  • Use digital imaging for densitometry

  • Analyze with appropriate software (ImageJ/FIJI)

• ELISA protocol:

  • Coat plates with capture antibody (1-10 μg/ml)

  • Block with 3-5% BSA

  • Apply samples and standards in duplicate

  • Detect with HRP-conjugated secondary antibody

  • Develop with TMB substrate

  • Read absorbance at 450 nm

• Protein normalization strategies:

  • Total protein normalization (REVERT or Ponceau staining)

  • Housekeeping proteins as loading controls

  • Spiked-in internal standards

Careful quantification is essential for detecting subtle changes in protein abundance, as seen in studies examining plant immune responses .

How can At5g19175 antibody be used in combination with other detection methods?

Multi-modal detection enhances experimental rigor:

• Antibody-fluorescent protein fusion complementation:

  • Validate GFP-tagged protein localization with antibody staining

  • Confirm fusion protein function through complementation assays

  • Use antibody for quantification of expression levels

• Antibody-mass spectrometry integration:

  • Immunoprecipitate with At5g19175 antibody

  • Identify post-translational modifications by mass spectrometry

  • Validate specific modifications with modification-specific antibodies

• Chromatin immunoprecipitation (ChIP) applications:

  • If At5g19175 has DNA-binding properties, perform ChIP

  • Optimize crosslinking conditions (1-3% formaldehyde, 10-20 minutes)

  • Sonicate to generate 200-500 bp fragments

  • Immunoprecipitate with At5g19175 antibody

  • Analyze by qPCR or sequencing

Combining methodologies provides stronger evidence for biological findings, as demonstrated in studies that used both biochemical fractionation and microscopy to determine protein localization .

What troubleshooting strategies are most effective for At5g19175 antibody experiments?

When encountering experimental issues:

Systematic troubleshooting is essential for antibody-based experiments. Document all optimization steps to ensure reproducibility across experiments .

How can At5g19175 antibody be evaluated for cross-reactivity with other plant species?

When extending antibody use to other species:

• Sequence comparison approach:

  • Align epitope sequence across species using BLAST

  • Calculate percent identity and similarity

  • Focus on species with >70% epitope conservation

• Empirical testing protocol:

  • Perform immunoblotting with protein extracts from multiple species

  • Start with higher antibody concentration (1:500)

  • Include Arabidopsis extract as positive control

  • Document cross-reactivity pattern

• Specificity confirmation:

  • Perform peptide competition assays

  • Test antibody on mutant/silenced plants when available

Many plant antibodies show cross-reactivity across species, as seen with the Anti-Lhcb2 antibody that reacts with proteins from multiple plant species including Arabidopsis, barley, and maize .

What modifications are needed for At5g19175 antibody use in crop species?

When adapting protocols for crop species:

• Extraction buffer modifications:

  • Increase buffer:tissue ratio for starchy tissues

  • Add PVP or PVPP (1-2%) to remove phenolics and tannins

  • Consider specific interfering compounds for each species

• Background reduction strategies:

  • Pre-adsorb antibody against plant extract lacking target protein

  • Optimize blocking conditions (try species-specific serums)

  • Increase wash stringency to reduce non-specific binding

• Signal optimization:

  • Consider signal amplification systems for low abundance targets

  • Test alternative secondary antibodies

  • Optimize incubation times and temperatures

Methodological adaptations are essential when transferring antibody applications across species, particularly when moving from model plants to crops with more complex tissue composition .

How can At5g19175 antibody be used to study protein dynamics during environmental stress?

To investigate stress-induced protein changes:

• Stress treatment design:

  • Apply controlled stress conditions (drought, salt, temperature)

  • Include appropriate time course (0, 1, 3, 6, 12, 24 hours)

  • Prepare parallel samples for transcript and protein analysis

• Protein dynamics assessment:

  • Monitor total protein levels by immunoblotting

  • Examine subcellular redistribution through fractionation

  • Assess complex formation changes via co-immunoprecipitation

• Post-translational modifications:

  • Use phospho-specific antibodies if available

  • Perform λ-phosphatase treatment to confirm phosphorylation

  • Consider mobility shift assays to detect modifications

Environmental stresses often trigger rapid protein modifications and redistribution, similar to the dynamic changes observed during immune responses in plants .

What approaches enable studying At5g19175 protein-DNA interactions?

If At5g19175 has DNA-binding properties:

• Chromatin immunoprecipitation protocol:

  • Crosslink tissue with 1% formaldehyde (10 minutes at room temperature)

  • Quench with 0.125 M glycine

  • Extract and shear chromatin to 200-500 bp fragments

  • Immunoprecipitate with At5g19175 antibody

  • Reverse crosslinks and purify DNA

  • Analyze by qPCR or sequencing

• In vitro DNA binding analysis:

  • Express recombinant protein

  • Perform electrophoretic mobility shift assay (EMSA)

  • Verify specificity with At5g19175 antibody supershift

  • Identify binding motifs through systematic analysis

These approaches have been successfully employed to study DNA-binding properties of plant transcription factors and regulatory proteins involved in stress and immune responses .