FLXL4 Antibody

FLXL4 Protein Background

What is FLXL4 protein in Arabidopsis thaliana?

FLXL4 (FLOWERING LOCUS C EXPRESSOR-LIKE 4) is a protein encoded by the Q9FH51 gene in Arabidopsis thaliana (Mouse-ear cress) . It belongs to a family of proteins involved in flowering regulation pathways. This protein plays a significant role in plant developmental processes, particularly in the transition from vegetative to reproductive growth. Understanding its expression patterns and interactions requires specific detection tools such as the FLXL4 Antibody.

Technical Specifications

What are the key specifications of the FLXL4 Antibody?

The FLXL4 Antibody (CSB-PA120362XA01DOA) is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana FLXL4 protein . It is supplied in liquid form with a storage buffer containing 0.03% Proclin 300 as a preservative and 50% Glycerol, 0.01M PBS at pH 7.4 . This antibody has been purified using antigen affinity methods and is specifically designed to react with Arabidopsis thaliana samples . It has been validated for ELISA and Western Blot applications, making it suitable for protein detection and quantification studies .

Storage and Stability

How should FLXL4 Antibody be stored to maintain optimal activity?

Upon receipt, FLXL4 Antibody should be stored at -20°C or -80°C to maintain its activity and specificity . Repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and reduce binding efficiency . For short-term use, small aliquots can be prepared to minimize freeze-thaw cycles. The 50% glycerol in the storage buffer helps maintain stability during freezing. Long-term stability studies suggest that proper storage can maintain antibody activity for up to 12 months, although batch-specific validation is recommended for critical experiments.

Validated Applications

What applications has the FLXL4 Antibody been validated for?

The FLXL4 Antibody has been specifically validated for ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blot (WB) applications for the identification of the antigen . These techniques allow researchers to detect and quantify FLXL4 protein in plant tissue extracts. While these are the validated applications, experienced researchers may adapt protocols for immunohistochemistry, immunoprecipitation, or chromatin immunoprecipitation after proper validation. Each application requires specific optimization steps to ensure reliable results.

Western Blot Methodology

What is the recommended Western Blot protocol for FLXL4 Antibody?

For Western Blot applications using FLXL4 Antibody, researchers should prepare protein extracts from Arabidopsis thaliana tissues using a buffer containing protease inhibitors to prevent degradation. After protein separation by SDS-PAGE and transfer to a membrane, blocking should be performed with 5% non-fat milk or BSA in TBST. The FLXL4 Antibody should be diluted in blocking buffer (typically 1:1000 to 1:5000, though optimization is recommended) and incubated overnight at 4°C. After washing with TBST, an appropriate secondary antibody (anti-rabbit IgG) conjugated with HRP or a fluorescent tag should be applied. Signal detection can be performed using chemiluminescence or fluorescence imaging systems. A validation step using positive and negative controls is essential for confirming specificity.

ELISA Applications

How should FLXL4 Antibody be used in ELISA experiments?

For ELISA applications, researchers can use the FLXL4 Antibody in either direct, indirect, sandwich, or competitive formats depending on experimental requirements. For indirect ELISA, coat plates with the antigen, block with appropriate buffer, then apply diluted FLXL4 Antibody (starting dilutions of 1:1000 to 1:5000 are recommended with optimization). After washing, apply HRP-conjugated secondary antibody and develop using an appropriate substrate. For quantitative analysis, standard curves should be established using purified recombinant FLXL4 protein. Cross-reactivity testing is advised when working with complex plant extracts to ensure specificity of detection.

Antibody Specificity Analysis

How can researchers verify the specificity of FLXL4 Antibody?

Verification of FLXL4 Antibody specificity is critical for research validity. Multiple approaches are recommended: (1) Parallel testing with FLXL4 knockout/knockdown plants as negative controls; (2) Pre-absorption tests with purified recombinant FLXL4 protein to demonstrate binding competition; (3) Mass spectrometry analysis of immunoprecipitated proteins to confirm identity; (4) Testing cross-reactivity with related proteins in the FLXL family; and (5) Comparison of results with alternative antibodies or detection methods. Multiple detection techniques (Western blot, ELISA, immunofluorescence) should yield consistent results if specificity is high. Researchers should document all validation steps in publications.

Cross-Reactivity Assessment

Does the FLXL4 Antibody cross-react with related proteins?

Troubleshooting Inconsistent Results

What factors might contribute to variable results when using FLXL4 Antibody?

Inconsistent results when using FLXL4 Antibody may stem from several factors:

• Sample preparation issues: Incomplete protein extraction, protein degradation, or post-translational modifications affecting epitope recognition

• Experimental conditions: Suboptimal antibody dilution, inappropriate blocking agents, or buffer composition

• Detection system limitations: Expired reagents, improper secondary antibody selection, or instrument calibration issues

• Biological variability: Developmental stage-dependent expression, tissue-specific regulation, or stress-induced changes

• Antibody lot variation: Differences in affinity or specificity between manufacturing batches

Systematic troubleshooting approaches include titrating antibody concentrations, modifying incubation conditions, testing alternative sample preparation methods, and including appropriate positive and negative controls in each experiment.

Experimental Controls

What controls should be included in experiments using FLXL4 Antibody?

Robust experimental design with FLXL4 Antibody requires several controls:

• Positive control: Wild-type Arabidopsis thaliana tissue with known FLXL4 expression

• Negative control: FLXL4 knockout/knockdown plant tissue or non-plant tissue

• Technical control: Primary antibody omission to assess non-specific binding of secondary antibody

• Loading control: Detection of housekeeping proteins (e.g., actin, tubulin) to normalize protein loading

• Specificity control: Pre-absorption of antibody with recombinant FLXL4 protein

• Cross-reactivity control: Testing against tissues expressing related FLXL family proteins

These controls help validate results and troubleshoot potential issues in the experimental workflow. Documentation of all controls is essential for publication and reproducibility.

Quantification Methods

How can researchers quantify FLXL4 protein levels accurately?

Accurate quantification of FLXL4 protein requires methodological rigor and appropriate analytical approaches:

For most applications, a combination of Western blot for qualitative analysis followed by ELISA for precise quantification provides complementary data. Signal normalization to total protein or housekeeping proteins is essential for comparative studies.

Statistical Analysis of FLXL4 Expression Data

What statistical approaches are recommended for analyzing FLXL4 expression data?

• Normality testing (Shapiro-Wilk or Kolmogorov-Smirnov) should be performed before selecting parametric or non-parametric tests

• For comparing two conditions, t-tests (parametric) or Mann-Whitney U tests (non-parametric) are appropriate

• For multiple conditions, ANOVA with post-hoc tests (Tukey's or Bonferroni) or Kruskal-Wallis tests are recommended

• Technical replicates should be averaged before statistical analysis of biological replicates

• Minimum sample size determination through power analysis ensures adequate statistical power

• Effect size calculations (Cohen's d or similar) help interpret biological significance beyond statistical significance

Data visualization using box plots or violin plots rather than simple bar graphs better represents data distribution and variability. Transparent reporting of all statistical methods, sample sizes, and outlier handling enhances reproducibility.

Protein-Protein Interaction Studies

How can FLXL4 Antibody be used to investigate protein-protein interactions?

FLXL4 Antibody enables investigation of protein-protein interactions through several techniques:

• Co-immunoprecipitation (Co-IP): Precipitate FLXL4 using the antibody and identify interacting proteins by mass spectrometry or Western blot

• Proximity ligation assay (PLA): Detect in situ interactions between FLXL4 and candidate interacting proteins

• Chromatin immunoprecipitation (ChIP): Identify DNA regions associated with FLXL4 if it functions in transcriptional complexes

• Pull-down assays: Use immobilized FLXL4 Antibody to isolate protein complexes from plant extracts

• Bimolecular fluorescence complementation (BiFC): Validate direct interactions identified through antibody-based methods

Optimization of buffer conditions is critical, as overly stringent conditions may disrupt weak interactions while insufficient stringency may result in non-specific binding. Cross-linking prior to extraction can stabilize transient interactions. Reciprocal verification (immunoprecipitating with antibodies against interacting proteins) strengthens confidence in identified interactions.

Subcellular Localization Studies

How can FLXL4 Antibody be used to determine subcellular localization?

Determining FLXL4 subcellular localization provides insight into protein function and regulatory mechanisms. The FLXL4 Antibody can be used in:

• Immunofluorescence microscopy: Fix and permeabilize plant cells/tissues, incubate with FLXL4 Antibody followed by fluorescently-labeled secondary antibody, and co-stain with organelle markers

• Immunogold electron microscopy: Greater resolution for precise subcellular localization

• Subcellular fractionation: Separate cellular compartments and detect FLXL4 by Western blot in each fraction

• Protease protection assays: Determine membrane topology of FLXL4 using protease digestion of intact organelles

• Nuclear/cytoplasmic extraction: Specifically assess partitioning between these compartments

Each method has distinct advantages, and complementary approaches provide stronger evidence for localization. Controls for fixation artifacts, extraction efficiency, and antibody specificity are essential for reliable interpretation.

Post-translational Modification Analysis

Can FLXL4 Antibody detect post-translationally modified forms of the protein?

The ability of FLXL4 Antibody to detect post-translationally modified forms depends on epitope location and modification type. Researchers investigating post-translational modifications (PTMs) should consider:

• Multiple band detection: Western blots showing bands at different molecular weights may indicate PTMs

• Phosphatase treatment: Compare samples before and after treatment to identify phosphorylated forms

• 2D gel electrophoresis: Separate proteins by isoelectric point and molecular weight to identify charge variants

• Lectin affinity: Enrich for glycosylated forms before immunodetection

• Mass spectrometry: Identify precise PTM types and locations after immunoprecipitation

Modifications near the epitope recognized by the antibody may reduce binding affinity, potentially causing false negatives. Generation of modification-specific antibodies may be necessary for comprehensive PTM studies of FLXL4 protein.

Evolutionary Conservation of FLXL4

How conserved is FLXL4 across plant species and can this antibody detect orthologs?

FLXL4 belongs to a plant-specific protein family with varying degrees of conservation across species. Sequence alignment analysis shows highest conservation among Brassicaceae family members, with decreasing similarity in monocots and other plant families. The FLXL4 Antibody was raised against Arabidopsis thaliana FLXL4 and is specifically validated for this species . Cross-reactivity with orthologs depends on epitope conservation. Testing in closely related species like Brassica may be successful, while detection in distant species requires validation. Researchers working with non-Arabidopsis species should perform preliminary cross-reactivity tests or consider developing species-specific antibodies. Computational prediction of epitope conservation can guide experimental design for cross-species applications.

Developmental Expression Patterns

How does FLXL4 expression change during plant development?

Comprehensive studies of FLXL4 expression throughout plant development require sampling across multiple tissues and developmental stages. The FLXL4 Antibody can be used to:

• Track temporal expression changes from seedling to mature plant stages

• Compare expression in vegetative versus reproductive tissues

• Analyze tissue-specific expression patterns through immunohistochemistry

• Correlate protein levels with developmental transitions

• Examine expression changes in response to environmental factors or stresses

Experimental design should include standardized sampling procedures, appropriate normalization controls, and consideration of diurnal or circadian regulation. Complementary approaches using transcriptomics can provide additional context for protein expression patterns detected with the antibody.

Integrating FLXL4 Antibody with Advanced Technologies

How can FLXL4 Antibody be integrated with emerging research technologies?

The FLXL4 Antibody can be adapted for use with several cutting-edge research technologies:

• Single-cell proteomics: Combining microfluidics with antibody-based detection

• Super-resolution microscopy: Nanoscale localization of FLXL4 within cellular compartments

• Spatial transcriptomics: Correlating protein detection with local gene expression patterns

• CRISPR-based genomic tagging: Validating endogenous tagging approaches

• Antibody engineering: Developing recombinant fragments for improved specificity or novel applications

These integrative approaches expand research possibilities beyond traditional applications, potentially revealing new aspects of FLXL4 biology and function. Methodological optimization and technological validation are essential when pioneering such applications.

Translational Research Potential

What is the potential for FLXL4 research to impact agricultural applications?

While FLXL4 Antibody is designated for research use only and not for diagnostic or therapeutic procedures , the fundamental knowledge generated may have broader implications:

• Understanding flowering time regulation mechanisms for crop improvement

• Identifying potential targets for breeding programs focusing on developmental traits

• Developing molecular markers for selection of favorable agricultural traits

• Engineering stress-responsive developmental pathways in crop species

• Discovering novel interaction networks that influence plant productivity

Researchers pursuing such translational directions should maintain focus on fundamental mechanisms while considering potential agricultural applications. Collaborations between academic researchers and plant breeding programs can accelerate knowledge transfer from basic research to practical applications.