At5g42235 Antibody

What is At5g42235 and what cellular functions does this protein perform in Arabidopsis thaliana?

At5g42235 is a protein encoded by the Arabidopsis thaliana genome, identified with UniProt number Q3E8I8 and Entrez Gene ID 2746178 . While the specific function of At5g42235 isn't directly stated in current literature, its study is important within the plant research community.

To determine the function of At5g42235, researchers should implement a multifaceted approach:

• Sequence analysis to identify conserved domains

• Expression studies across different tissues and developmental stages

• Generation of knockout/knockdown mutants to observe phenotypic changes

• Protein localization studies to determine subcellular location

• Interaction studies to identify binding partners

Based on research with similar plant proteins, At5g42235 may be involved in processes such as autophagy (similar to ATG proteins) , stress response mechanisms (like ABCG transporters) , or plant development pathways.

What validation methods should be used to confirm the specificity of At5g42235 antibody?

Multiple validation methods should be employed to confirm antibody specificity, following the principles outlined in enhanced validation frameworks :

• Orthogonal validation: Compare protein levels determined by the At5g42235 antibody with measurements from antibody-independent methods (e.g., mass spectrometry) across samples with varying expression. Strong correlation (R>0.5) supports specificity.

• Genetic validation: Use genetic knockdown approaches (RNAi or CRISPR-Cas9) to reduce expression of At5g42235. A corresponding reduction in antibody signal confirms specificity.

• Independent antibody validation: Compare staining patterns from multiple antibodies raised against different epitopes of At5g42235.

• Recombinant expression validation: Express tagged recombinant At5g42235 protein and confirm antibody detection. Current commercial antibodies have been confirmed to recognize recombinant At5g42235 .

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to identify all proteins pulled down, confirming enrichment of At5g42235 peptides.

Vendor information indicates that current antibodies recognize recombinant At5g42235 but "reactivity on endogenous protein needs to be confirmed" , highlighting the importance of validation with endogenous protein sources.

What are the recommended applications for At5g42235 antibody?

Based on vendor specifications, At5g42235 antibody is validated for :

• ELISA (Enzyme-Linked Immunosorbent Assay): For detecting and quantifying the protein in solution.

• Western Blot (WB): For detecting the protein in tissue homogenates or extracts.

  • Recommended dilution: 1:1000

Additional potential applications requiring further validation include:

Each application requires independent validation following the principles outlined in enhanced validation frameworks .

How should At5g42235 antibody be stored and reconstituted for optimal performance?

Proper storage and handling are critical for maintaining antibody performance:

Storage conditions :

• Store lyophilized antibody at -20°C

• Once reconstituted, make small aliquots to avoid repeated freeze-thaw cycles

• For long-term storage, keep at -20°C or -80°C

Reconstitution protocol :

• Centrifuge the antibody vial briefly before opening to collect material from cap/walls

• Add 50 μl of sterile water to the lyophilized antibody

• Mix gently by inversion, avoid vortexing which can cause protein denaturation

• Allow to stand at room temperature for 5 minutes before aliquoting

Additional considerations:

• Keep antibody solutions on ice when working at the bench

• Avoid contamination by using sterile technique

• For long-term storage of reconstituted solutions, consider adding preservatives (e.g., 0.02% sodium azide)

• Record date of reconstitution and track number of freeze-thaw cycles

What controls should be included when using At5g42235 antibody in Western blot experiments?

Robust experimental design requires appropriate controls:

Essential controls :

• Positive controls:

  • Recombinant At5g42235 protein (provided as 200μg antigen with antibody)

  • Wild-type Arabidopsis tissue expressing At5g42235

• Negative controls:

  • Pre-immune serum (provided as 1ml with antibody)

  • Tissue with low/no expression of At5g42235

  • Secondary antibody only (omit primary antibody)

• Loading controls:

  • Antibodies against housekeeping proteins (actin, tubulin, GAPDH)

  • Total protein staining (Ponceau S, Coomassie)

Additional validation controls:

• Blocking peptide: Pre-incubate antibody with excess target peptide

• Molecular weight markers: Confirm band at expected molecular weight

• Protein concentration gradient: Demonstrate signal proportionality

• Technical replicates: Minimum of three independent experiments

How can orthogonal validation methods be applied to confirm At5g42235 antibody specificity?

Orthogonal validation requires comparing antibody-based detection with antibody-independent methods :

Transcriptomics-Proteomics Correlation:

• Select 6-10 Arabidopsis samples (different tissues, developmental stages, stress conditions) with varying At5g42235 expression

• Quantify protein levels using Western blot or ELISA with At5g42235 antibody

• Measure mRNA levels by RT-qPCR or RNA-seq

• Calculate correlation coefficient between protein and mRNA levels

Mass Spectrometry Validation:

• Perform relative quantification of At5g42235 across the same sample panel using MS-based proteomics

• Compare MS-based quantification with antibody-based detection

• Calculate correlation coefficient (R>0.5 confirms specificity)

Sample Preparation Optimization:

• Buffer composition: Test different extraction buffers

  • Standard RIPA buffer

  • Plant-specific buffers with higher detergent concentrations

  • Buffers with plant protease inhibitor cocktails

• Tissue disruption methods:

  • Mortar and pestle with liquid nitrogen

  • Bead beating with different bead types

  • Sonication parameters (time, amplitude)

• Protein extraction efficiency:

  • TCA/acetone precipitation

  • Phenol extraction

  • Commercial plant protein extraction kits

Western Blot Protocol Optimization:

• Blocking conditions: Test various agents

  • 5% non-fat dry milk

  • 3-5% BSA

  • Commercial plant-optimized blocking reagents

• Antibody parameters:

  • Concentration titration (1:500 to 1:2000)

  • Incubation temperature (4°C vs. room temperature)

  • Incubation time (2 hours vs. overnight)

• For difficult samples:

  • Epitope retrieval methods

  • Pre-absorption with plant powder

  • Subcellular fractionation to enrich target

As emphasized in validation literature: "antibodies must be validated in an application-specific manner" , requiring optimization protocols tailored to specific sample types.

How does the polyclonal nature of At5g42235 antibody impact experimental reproducibility?

The polyclonal nature of the At5g42235 antibody has significant implications for reproducibility:

Characteristics affecting reproducibility:

• Multiple epitope recognition:

  • Advantage: More robust detection across experimental conditions

  • Challenge: Potential recognition of shared epitopes on related proteins

• Lot-to-lot variability:

  • Different production batches may have varying:

    • Epitope affinities

    • Sensitivity levels

    • Background patterns

Impact on quantitative analysis:

Strategies to enhance reproducibility:

• Detailed reporting:

  • Document antibody catalog number, lot number, dilution

  • Report all validation steps for your specific application

• Standardized validation :

  • Implement orthogonal validation for each new lot

  • Include consistent positive and negative controls

• Complementary approaches:

  • Confirm key findings with independent methods

  • Validate with different antibodies against the same target

What approaches are recommended for quantitative analysis of At5g42235 protein expression?

For rigorous quantitative analysis:

Western Blot Quantification:

• Quantitative optimization:

  • Establish linear dynamic range with recombinant standards

  • Determine detection threshold and saturation point

  • Use gradient loading to confirm signal proportionality

• Signal analysis:

  • Use digital image acquisition systems

  • Apply appropriate background subtraction

  • Normalize to validated loading controls

• Statistical analysis:

  • Perform minimum three biological replicates

  • Calculate coefficient of variation

  • Apply appropriate statistical tests

ELISA-Based Quantification:

• Standard curve development:

  • Use recombinant At5g42235 for absolute quantification

  • Ensure curve covers physiological range

• Sample considerations:

  • Standardize extraction across tissues

  • Validate matrix effects

  • Include spike-in recovery controls

Immunoprecipitation-Based Approaches:

• Standard Co-Immunoprecipitation (Co-IP):

  • Use At5g42235 antibody to pull down protein complexes

  • Analyze co-precipitated proteins by Western blot or mass spectrometry

  • Include controls (pre-immune serum, IgG control)

• IP-Mass Spectrometry :

  • Immunoprecipitate with At5g42235 antibody

  • Identify all co-precipitated proteins by MS

  • Filter against appropriate negative controls

Proximity-Based Methods:

• Proximity Ligation Assay (PLA):

  • Combine At5g42235 antibody with antibodies against suspected partners

  • Visualize interactions as fluorescent spots

  • Quantify interaction signals in different cellular compartments

• Validation experiments:

  • Competitive peptide approaches

  • Domain mapping with truncated proteins

  • Genetic knockout validation

Integration with functional studies:

As emphasized in antibody validation literature, "a technique for isolation and concentration of specific proteins from complex mixtures" like immunoprecipitation combined with mass spectrometry provides powerful insight into protein interactions when properly controlled.