At5g37474 Antibody

What is At5g37474 Antibody and what target does it recognize?

At5g37474 Antibody (CSB-PA651195XA01DOA) is a polyclonal antibody raised in rabbits against a recombinant Arabidopsis thaliana At5g37474 protein. The antibody specifically recognizes the At5g37474 target protein in Arabidopsis thaliana (Mouse-ear cress), which is a widely used model organism in plant molecular biology research. The antibody is generated through antigen affinity purification methods to ensure specificity for the target protein .

As a polyclonal preparation, this antibody contains a heterogeneous mixture of immunoglobulins that recognize multiple epitopes on the target antigen, potentially providing robust detection capabilities across different experimental conditions. The antibody is specifically designed for research applications only and is not validated for diagnostic or therapeutic procedures .

What are the validated applications for At5g37474 Antibody?

At5g37474 Antibody has been validated for two primary research applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): The antibody can be used in various ELISA formats to detect and quantify At5g37474 protein in research samples.

• Western Blotting (WB): The antibody effectively detects At5g37474 protein in Western blot applications, which are crucial for identifying protein expression, modifications, and molecular weight .

When designing experiments, researchers should note that validation has been specifically performed for these two applications. While the antibody might work in other immunological techniques, additional optimization and validation would be required if adapting it for applications beyond ELISA and Western blotting. Methodology for each application may need to be customized according to sample type and experimental conditions.

What are the optimal storage conditions for maintaining At5g37474 Antibody activity?

For optimal preservation of At5g37474 Antibody activity, researchers should follow these evidence-based storage protocols:

• Upon receipt, store the antibody at either -20°C or -80°C for long-term preservation

• Avoid repeated freeze-thaw cycles, as these can significantly degrade antibody performance

• The antibody is supplied in liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

• Working aliquots can be prepared to minimize freeze-thaw cycles

Research has demonstrated that proper storage conditions directly impact antibody performance in downstream applications. For instance, antibodies stored according to manufacturer recommendations typically maintain >90% of their binding activity compared to significant reductions observed with improper storage protocols or excessive freeze-thaw cycles.

How should I optimize Western blot protocols for At5g37474 Antibody?

When optimizing Western blot protocols for At5g37474 Antibody, researchers should implement the following methodological approach:

• Sample Preparation:

  • Extract total protein from Arabidopsis thaliana tissues using standard extraction buffers (RIPA or similar)

  • Include protease inhibitors to prevent degradation

  • Determine protein concentration using Bradford or BCA assays

• Gel Electrophoresis:

  • Load 20-50 μg of protein per lane (optimize based on target abundance)

  • Use 10-12% SDS-PAGE gels for optimal separation

• Transfer and Blocking:

  • Transfer proteins to PVDF or nitrocellulose membranes

  • Block with 5% non-fat dry milk or 3-5% BSA in TBST for 1 hour at room temperature

• Antibody Incubation:

  • Dilute At5g37474 Antibody at 1:500 to 1:2000 (determine optimal dilution empirically)

  • Incubate overnight at 4°C

  • Wash 3-5 times with TBST buffer

• Detection:

  • Use appropriate HRP-conjugated secondary anti-rabbit antibody

  • Visualize using chemiluminescence detection systems

Western blot optimization is critical as this antibody is specifically validated for identification of the target antigen. Troubleshooting may be necessary if non-specific bands appear or if signal strength is insufficient.

What controls should be included when using At5g37474 Antibody in immunological assays?

Including appropriate controls is essential for robust experimental design when using At5g37474 Antibody:

Essential Controls:

• Positive Control:

  • Arabidopsis thaliana tissue/cell lysate known to express At5g37474 protein

  • Recombinant At5g37474 protein (when available)

• Negative Control:

  • Tissue/cells from knockout or knockdown plants lacking At5g37474 expression

  • Non-plant samples or irrelevant protein samples

• Antibody Controls:

  • Primary antibody omission control (to detect non-specific binding of secondary antibody)

  • Pre-immune serum control (where available)

  • Isotype control (rabbit IgG at equivalent concentration)

• Loading Control:

  • Detection of housekeeping proteins (e.g., actin, tubulin, GAPDH) to ensure equal loading and transfer efficiency

Control Implementation Table:

These controls help validate experimental results and troubleshoot potential issues in immunoassays .

What is the recommended ELISA protocol for At5g37474 Antibody?

For optimal ELISA performance using At5g37474 Antibody, researchers should follow this methodological approach:

• Plate Preparation:

  • Coat 96-well plates with capture antibody or target antigen extract (for indirect ELISA)

  • Use 50-100 μl per well of coating buffer (typically carbonate/bicarbonate buffer, pH 9.6)

  • Incubate overnight at 4°C

• Blocking:

  • Block with 200-300 μl of 1-5% BSA in PBS or appropriate blocking buffer

  • Incubate for 1-2 hours at room temperature

• Sample Addition:

  • Add samples and standards in appropriate dilutions

  • Incubate for 1-2 hours at room temperature

• Primary Antibody Incubation:

  • Add At5g37474 Antibody diluted 1:500 to 1:2000 in antibody diluent

  • Incubate for 1-2 hours at room temperature or overnight at 4°C

• Secondary Antibody Incubation:

  • Add HRP-conjugated anti-rabbit secondary antibody at recommended dilution

  • Incubate for 1 hour at room temperature

• Detection:

  • Add appropriate substrate (TMB, ABTS)

  • Measure absorbance at recommended wavelength

• Analysis:

  • Generate standard curve from known concentration samples

  • Calculate concentration of target protein in test samples

Wash thoroughly with PBST or TBST between each step, typically 3-5 washes. This protocol should be optimized based on sample type and experimental conditions.

How can I address specificity issues with At5g37474 Antibody?

When confronting specificity issues with At5g37474 Antibody, implement these methodological approaches:

• Epitope Competition Assay:

  • Pre-incubate the antibody with excess purified target protein

  • Run parallel assays with competed and non-competed antibody

  • Specific signal should be significantly reduced in the competed sample

• Gradient Optimization:

  • Test a range of antibody dilutions (1:250 to 1:5000)

  • Identify the optimal concentration that maximizes specific signal while minimizing background

• Buffer Optimization:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Adjust detergent concentrations in wash buffers (0.05% to 0.1% Tween-20)

  • Consider including carrier proteins in antibody diluent

• Antigen Retrieval (for tissue samples):

  • Test different antigen retrieval methods for tissue sections

  • Compare heat-induced vs. enzymatic retrieval methods

• Knockout/Knockdown Validation:

  • Compare signal between wild-type and At5g37474 knockout/knockdown samples

  • True specific signal should be absent or significantly reduced in knockout samples

Remember that as a polyclonal antibody, At5g37474 Antibody recognizes multiple epitopes, which can increase sensitivity but potentially introduce more non-specific binding compared to monoclonal antibodies.

What factors might affect reproducibility when using At5g37474 Antibody?

Several experimental factors can impact the reproducibility of results when using At5g37474 Antibody:

• Antibody Lot-to-Lot Variation:

  • Being a polyclonal antibody, different lots may have variations in epitope recognition patterns

  • Validate new lots against previous experimental conditions

  • Consider creating reference samples for inter-lot comparison

• Sample Preparation Inconsistencies:

  • Variations in protein extraction efficiency

  • Protein degradation during sample processing

  • Inconsistent sample concentration determination

• Experimental Condition Variations:

  • Temperature fluctuations during incubation steps

  • Inconsistent washing procedures

  • Variations in detection reagent activity

• Antibody Storage and Handling:

  • Repeated freeze-thaw cycles degrading antibody quality

  • Improper temperature storage

  • Bacterial contamination

• Cross-Reactivity Considerations:

  • Similar epitopes in related proteins

  • Non-specific binding due to high antibody concentration

Reproducibility Enhancement Table:

Implementing these strategies will help ensure consistent and reliable results across experiments .

How can I determine the optimal concentration of At5g37474 Antibody for my experiments?

To determine the optimal concentration of At5g37474 Antibody for your specific experimental setup, employ this methodological titration approach:

• Serial Dilution Testing:

  • Prepare a range of antibody dilutions (e.g., 1:100, 1:500, 1:1000, 1:2000, 1:5000)

  • Run parallel assays using identical samples and conditions

  • Compare signal-to-noise ratio across dilutions

• Signal Intensity Analysis:

  • Plot signal intensity versus antibody concentration

  • Identify the dilution that provides maximum specific signal while minimizing background

• Comparative Sample Analysis:

  • Test the antibody performance on samples with known high, medium, and low levels of target expression

  • Ensure the selected concentration can detect the target across the expected expression range

• Antibody Consumption Optimization:

  • Consider the balance between optimal signal and antibody consumption

  • Higher concentrations may provide stronger signals but consume more antibody

Sample Titration Results Table:

The optimal concentration will depend on your specific application, sample type, and detection method. For Western blotting, a starting dilution of 1:1000 is often recommended, while for ELISA, dilutions between 1:500 and 1:2000 are typically tested .

How can At5g37474 Antibody be used in studying protein-protein interactions?

For investigating protein-protein interactions involving At5g37474 protein, researchers can implement these advanced methodological approaches:

• Co-Immunoprecipitation (Co-IP):

  • Use At5g37474 Antibody coupled to Protein A/G beads

  • Incubate with Arabidopsis thaliana lysates

  • Elute and analyze co-precipitated proteins via mass spectrometry or Western blotting

  • Implementation protocol:
    a. Couple 2-5 μg of At5g37474 Antibody to 50 μl of Protein A/G beads
    b. Incubate with 500-1000 μg of total protein lysate
    c. Wash stringently to remove non-specific interactions
    d. Elute bound proteins and analyze by SDS-PAGE followed by Western blotting or mass spectrometry

• Proximity Ligation Assay (PLA):

  • Combine At5g37474 Antibody with antibodies against suspected interaction partners

  • Visualize protein-protein interactions in situ with cellular spatial resolution

  • Quantify interaction signals using appropriate imaging software

• Pull-down Assays with Recombinant Proteins:

  • Express recombinant At5g37474 as a tagged fusion protein

  • Use At5g37474 Antibody to validate expression and purification

  • Perform pull-down assays with plant lysates to identify interacting proteins

• Chromatin Immunoprecipitation (ChIP) (if At5g37474 has DNA-binding properties):

  • Use At5g37474 Antibody to precipitate protein-DNA complexes

  • Analyze bound DNA sequences through sequencing or qPCR

  • Identify potential regulatory targets

These advanced applications extend beyond basic detection and enable functional characterization of At5g37474 protein in complex biological systems. Optimization for each technique will be required as the antibody is primarily validated for ELISA and Western blotting.

What considerations should be made when using At5g37474 Antibody for quantitative analyses?

When utilizing At5g37474 Antibody for quantitative analyses, researchers should address these methodological considerations:

• Standard Curve Development:

  • Generate a standard curve using purified recombinant At5g37474 protein

  • Ensure linearity across the expected concentration range

  • Calculate the limit of detection (LOD) and limit of quantification (LOQ)

• Antibody Binding Kinetics Assessment:

  • Determine the binding affinity (Kd) of the antibody to its target

  • Assess potential high-dose hook effects in highly concentrated samples

  • Optimize incubation times to ensure equilibrium binding

• Signal Calibration Methods:

  • Implement internal calibration standards in each experiment

  • Use relative quantification with reference proteins when absolute standards are unavailable

  • Normalize signal to total protein or housekeeping proteins

• Technical Replication Strategy:

  • Perform a minimum of three technical replicates per sample

  • Calculate coefficient of variation (CV) between replicates (aim for CV <15%)

  • Implement statistical analysis appropriate for the experimental design

Quantitative Performance Metrics Table:

Understanding these quantitative parameters ensures reliable and reproducible quantification of At5g37474 protein across different experimental conditions and sample types .

How can At5g37474 Antibody be adapted for imaging applications?

While At5g37474 Antibody is primarily validated for ELISA and Western blotting, researchers can adapt it for various imaging applications through these methodological approaches:

• Immunofluorescence Microscopy:

  • Fixation Optimization:

    • Test multiple fixation methods (4% paraformaldehyde, methanol, or acetone)

    • Optimize fixation duration (10-30 minutes)

  • Permeabilization Protocol:

    • Compare detergents (0.1-0.5% Triton X-100, 0.1% Saponin)

    • Adjust permeabilization time (5-15 minutes)

  • Antibody Incubation:

    • Test higher concentrations than used for Western blotting (1:50 to 1:500)

    • Extend incubation times (overnight at 4°C)

  • Detection Enhancement:

    • Implement tyramide signal amplification for low-abundance targets

    • Use high-sensitivity fluorophore-conjugated secondary antibodies

• Immunohistochemistry (IHC):

  • Tissue Preparation:

    • Optimize fixation and embedding protocols for plant tissues

    • Test various section thicknesses (5-10 μm)

  • Antigen Retrieval:

    • Compare heat-induced retrieval methods (citrate buffer, pH 6.0)

    • Test enzymatic retrieval approaches (proteinase K)

  • Signal Development:

    • Evaluate chromogenic substrates (DAB, AEC)

    • Optimize development times

• Super-Resolution Microscopy Adaptation:

  • Use secondary antibodies conjugated to photoswitchable fluorophores

  • Implement protocol modifications specific to STED, STORM, or PALM techniques

  • Validate resolution improvement compared to conventional microscopy

Imaging Application Optimization Matrix:

These adaptations require additional validation steps since the antibody is not specifically tested for imaging applications in the provided information.

How can I validate At5g37474 Antibody specificity for my particular plant model?

For validating At5g37474 Antibody specificity in plant models beyond Arabidopsis thaliana, employ this methodological validation framework:

• Sequence Homology Analysis:

  • Perform sequence alignment between Arabidopsis At5g37474 and homologous proteins in your plant model

  • Calculate percent identity and similarity in the immunogen region

  • Proteins with >70% sequence identity in epitope regions have higher cross-reactivity potential

• Knockout/Knockdown Validation:

  • Test antibody on samples from plants with genetic modifications affecting At5g37474 homologs

  • Compare signal patterns between wild-type and modified plants

  • Expected result: Reduced or absent signal in knockout/knockdown samples

• Peptide Competition Assay:

  • Pre-incubate antibody with excess peptide/protein from your model plant

  • Run parallel Western blots with competed and non-competed antibody

  • Specific signals should be eliminated or significantly reduced in the competed sample

• Mass Spectrometry Confirmation:

  • Immunoprecipitate target protein using At5g37474 Antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm identity of captured proteins matches expected target

Validation Scoring System:

Complete validation typically requires multiple approaches, with greater confidence when multiple methods show consistent results .

What are the critical parameters for validating batch-to-batch consistency of At5g37474 Antibody?

For ensuring batch-to-batch consistency when working with At5g37474 Antibody over extended research periods, implement these methodological validation approaches:

• Reference Sample Testing:

  • Maintain aliquots of reference samples from initial experiments

  • Test each new antibody lot against these standards

  • Compare signal intensity, pattern, and background levels

• Quantitative Performance Metrics:

  • Determine EC50/IC50 values for each lot in standardized ELISA

  • Calculate and compare antibody titer across lots

  • Assess detection limit consistency

• Epitope Recognition Profile:

  • Perform epitope mapping using peptide arrays if available

  • Compare epitope recognition patterns between lots

  • Analyze recognition of primary vs. secondary epitopes

• Sensitivity and Specificity Assessment:

  • Test serial dilutions of target protein

  • Evaluate cross-reactivity with related proteins

  • Compare non-specific binding profiles

Batch Validation Documentation Table:

Establishing these validation parameters helps maintain experimental consistency throughout long-term research projects, especially important for polyclonal antibodies like At5g37474 Antibody that may have inherent lot-to-lot variations .

What methodological considerations should be made when evaluating potential cross-reactivity with related proteins?

When evaluating potential cross-reactivity of At5g37474 Antibody with related proteins, implement this structured methodological approach:

• Bioinformatic Prediction:

  • Identify proteins with sequence homology to At5g37474

  • Focus on proteins with >50% sequence identity in immunogen region

  • Analyze predicted epitopes using tools like BepiPred or DiscoTope

• Recombinant Protein Panel Testing:

  • Express recombinant versions of related proteins

  • Perform side-by-side Western blot analysis

  • Quantify relative binding affinity to each protein

• Knockout/Knockdown Array Analysis:

  • Test antibody on samples from plants with knockouts of At5g37474 and related genes

  • Create a cross-reactivity matrix based on signal presence/absence

  • Identify potential false positives

• Absorption Controls:

  • Pre-absorb antibody with recombinant related proteins

  • Compare signal before and after absorption

  • Quantify percent signal reduction as measure of cross-reactivity

Cross-Reactivity Evaluation Matrix:

This systematic approach allows researchers to identify potential cross-reacting proteins and implement appropriate controls in their experimental design. For research where absolute specificity is critical, additional validation steps or alternative detection methods may be warranted .