At1g28600 Antibody

What is At1g28600 and what role does it play in Arabidopsis thaliana?

At1g28600 refers to a specific gene in Arabidopsis thaliana (Mouse-ear cress) following the standard Arabidopsis genome annotation nomenclature. The "At" indicates Arabidopsis thaliana, "1" refers to chromosome 1, and "g28600" indicates it is the 28,600th gene on that chromosome. The protein encoded by this gene is identified in the UniProt database as Q94F40, though its specific biological functions require further characterization. Understanding this protein's expression patterns and localization is crucial for plant molecular biology research, particularly in studies of Arabidopsis development and stress responses. The antibody against this protein serves as a valuable tool for investigating its expression, localization, and potential interactions in the plant system .

What are the proper storage and handling conditions for At1g28600 antibody?

At1g28600 antibody requires careful storage and handling to maintain its integrity and functionality. Upon receipt, the antibody should be stored at either -20°C or -80°C. It is particularly important to avoid repeated freeze-thaw cycles, as these can significantly degrade antibody quality and reduce binding efficacy. The antibody is supplied in liquid form with a specific storage buffer composition of 50% Glycerol, 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative . When working with the antibody, standard protein handling protocols should be followed, including using sterile, nuclease-free tubes and filtered pipette tips. For long-term storage stability, it is recommended to aliquot the antibody into smaller volumes before freezing to minimize the number of freeze-thaw cycles. Working on ice during experimental procedures will help maintain antibody stability and activity .

What are the validated applications for At1g28600 antibody?

According to the product information, At1g28600 antibody has been specifically validated for two primary applications: Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) . These validated applications make the antibody suitable for both qualitative and quantitative protein analysis. In Western blot applications, the antibody allows researchers to detect the presence and molecular weight of At1g28600 protein in plant extracts, enabling comparisons of expression levels across different tissues, developmental stages, or experimental conditions. For ELISA applications, the antibody provides more precise quantitative measurements of At1g28600 protein levels, allowing for high-throughput analysis and sensitive detection even with small sample volumes. While these are the manufacturer-validated applications, researchers might also consider testing the antibody for other applications such as immunoprecipitation, immunohistochemistry, or immunofluorescence, though these would require additional validation .

What distinguishes polyclonal from monoclonal antibodies for At1g28600 detection?

The At1g28600 antibody described in the product information is a polyclonal antibody raised in rabbits . Understanding the differences between polyclonal and monoclonal antibodies is important for selecting the appropriate reagent for specific research applications:

What are effective validation strategies to ensure At1g28600 antibody specificity?

Comprehensive validation of At1g28600 antibody specificity is essential for producing reliable research results. A multi-faceted approach should be implemented:

• Genetic Controls: The most definitive validation approach involves comparing antibody signals between wild-type Arabidopsis and At1g28600 knockout/knockdown lines. A specific antibody should show significantly reduced or absent signal in genetic lines where the target protein is absent or reduced . This approach provides strong evidence that the antibody is truly detecting the intended target.

• Molecular Weight Verification: The detected protein band should match the predicted molecular weight of At1g28600. Significant deviations might indicate cross-reactivity or post-translational modifications.

• Peptide Competition Assay: Pre-incubating the antibody with excess purified At1g28600 protein or the immunizing peptide should substantially reduce specific signals in subsequent assays. This test confirms that the antibody is binding to the intended epitope rather than interacting non-specifically.

• Orthogonal Detection Methods: Comparing antibody-based detection with independent methods such as mass spectrometry or fluorescently tagged proteins provides additional confidence in specificity.

• Validation Scoring System:

These validation approaches should be documented and reported in research publications to enable proper evaluation of experimental results and reproducibility .

How can At1g28600 antibody be employed in protein-protein interaction studies?

The At1g28600 antibody can serve as a valuable tool for investigating protein-protein interactions through several methodological approaches:

• Co-Immunoprecipitation (Co-IP): This is the most direct application, where At1g28600 antibody is used to pull down the target protein along with its interacting partners from plant lysates. The protocol involves:

  • Preparing plant lysates under non-denaturing conditions to preserve protein complexes

  • Incubating with At1g28600 antibody to form antibody-protein complexes

  • Capturing these complexes using Protein A or G beads

  • Washing to remove non-specifically bound proteins

  • Eluting and analyzing the captured proteins by mass spectrometry or Western blot

• Proximity Ligation Assay (PLA): This technique allows visualization of protein interactions in situ with high sensitivity. It requires the At1g28600 antibody to be used alongside antibodies against potential interacting partners. When the proteins are in close proximity (within 40 nm), a signal is generated, providing spatial information about the interaction within the cell.

• Pull-down Validation: After identifying candidate interacting proteins through proteomic approaches, the At1g28600 antibody can be used in Western blot analysis to confirm the presence of specific partners in immunoprecipitated complexes .

• Reciprocal Verification: For confident identification of interaction partners, reciprocal co-IPs should be performed where antibodies against the potential interacting proteins are used to confirm the presence of At1g28600 in the pulled-down complexes.

When conducting these experiments, researchers must include appropriate controls:

• IgG control immunoprecipitations to identify non-specific binding

• Input samples to confirm the presence of proteins in the starting material

• Validation in At1g28600 knockout/knockdown lines when available

• Competing peptide controls to demonstrate specificity of interactions

These methodological approaches can provide valuable insights into the functional relationships of At1g28600 with other proteins in plant cellular processes .

What considerations are important when adapting At1g28600 antibody for imaging applications?

While the At1g28600 antibody has been validated for ELISA and Western blot applications , researchers may wish to adapt it for imaging techniques to study protein localization. This adaptation requires careful optimization and validation:

• Fixation Optimization: The fixation method significantly impacts epitope preservation and antibody accessibility. A comparison of different fixation protocols is recommended:

• Permeabilization: Optimization of detergent type and concentration is crucial for antibody access while preserving cellular structures. Typical options include Triton X-100 (0.1-0.5%), Tween-20 (0.05-0.2%), or saponin (0.1-0.5%) for membrane permeabilization.

• Antibody Dilution: Starting with a higher concentration (1:50 to 1:100) than used for Western blotting is typically recommended, with subsequent optimization based on signal-to-noise ratio.

• Signal Amplification: For low-abundance proteins, consider using signal amplification systems such as tyramide signal amplification or quantum dots for enhanced sensitivity.

• Validation Controls:

  • Negative controls: Primary antibody omission and staining in At1g28600 knockout lines

  • Competitive inhibition: Pre-absorption with immunizing peptide

  • Positive controls: Co-localization with known organelle markers

• Advanced Imaging Approaches:

  • Confocal microscopy for high-resolution subcellular localization

  • Super-resolution techniques (STORM, PALM, SIM) for nanoscale distribution

  • Electron microscopy with immunogold labeling for ultrastructural localization

Researchers should systematically optimize and validate each step when adapting the At1g28600 antibody for imaging applications, documenting the optimization process for reproducibility.

What strategies help overcome cross-reactivity challenges in non-Arabidopsis species?

When using the At1g28600 antibody in species other than Arabidopsis thaliana, researchers must address potential cross-reactivity challenges through systematic validation and optimization:

• Sequence Homology Analysis: Before experimental testing, perform bioinformatic analysis comparing At1g28600 sequences between Arabidopsis and the target species. Higher sequence similarity in the regions containing the epitopes increases the likelihood of successful cross-reactivity.

• Cross-Reactivity Probability Assessment:

• Western Blot Validation: The most direct approach to test cross-reactivity involves performing Western blot analysis on protein extracts from both Arabidopsis (positive control) and the target species. Compare band patterns, molecular weights, and signal intensities to assess specificity.

• Peptide Competition: Perform peptide competition assays with the immunizing peptide to confirm that any observed signals in the non-Arabidopsis species are due to specific binding rather than cross-reactivity with unrelated proteins.

• Immunoprecipitation-Mass Spectrometry: For definitive identification, immunoprecipitate the protein from the non-Arabidopsis species and analyze by mass spectrometry to confirm its identity as the true ortholog of At1g28600.

• Antibody Dilution Optimization: Cross-reactive antibodies often require different working dilutions in different species. Perform titration experiments to identify the optimal antibody concentration that maximizes specific signal while minimizing background .

• Extraction Buffer Adaptation: Modify protein extraction protocols to account for species-specific differences in tissue composition, such as adjusting detergent concentrations or adding compounds to remove interfering molecules like phenolics or polysaccharides.

These approaches help researchers determine whether the At1g28600 antibody can be reliably used in non-Arabidopsis species and establish the appropriate experimental conditions for such applications.

How does the immunogen selection affect At1g28600 antibody performance?

The choice of immunogen used to generate an antibody significantly impacts its performance characteristics and application suitability. For the At1g28600 antibody, a recombinant Arabidopsis thaliana At1g28600 protein was used as the immunogen . This selection has important implications:

• Immunogen Type Comparison:

• Epitope Accessibility: The use of a full recombinant protein as immunogen likely generated antibodies recognizing multiple epitopes across the protein, some of which may be more accessible in native versus denatured states. This affects application suitability, with some epitopes being more accessible in Western blot (denatured proteins) versus immunoprecipitation (native proteins).

• Post-Translational Modifications (PTMs): Standard recombinant protein production in bacterial systems does not incorporate PTMs that may be present in plant-expressed At1g28600. If the protein undergoes significant post-translational modification in plants, the antibody may show differential recognition of modified versus unmodified forms.

• Production System Effects: The recombinant protein immunogen was likely produced in a bacterial expression system, which can affect protein folding compared to the native plant protein. This may impact antibody recognition of conformational epitopes.

• Antigen Affinity Purification: The At1g28600 antibody underwent antigen affinity purification , which enhances specificity by selecting only those antibody molecules that bind to the target protein. This purification method generally improves performance compared to crude serum or protein A/G purification alone.

Understanding these immunogen-related factors helps researchers interpret experimental results and troubleshoot potential issues when working with the At1g28600 antibody .

What is the optimal Western blot protocol for At1g28600 detection?

Based on the antibody characteristics and general best practices for plant protein analysis, the following optimized Western blot protocol is recommended for At1g28600 detection:

Sample Preparation:

• Harvest and flash-freeze Arabidopsis tissue in liquid nitrogen

• Grind to a fine powder using mortar and pestle

• Extract proteins using extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1 mM DTT, protease inhibitor cocktail)

• Centrifuge at 14,000 × g for 15 minutes at 4°C

• Collect supernatant and determine protein concentration

Gel Electrophoresis and Transfer:

• Load 20-40 μg total protein per lane alongside molecular weight markers

• Separate proteins on 10-12% SDS-PAGE gel

• Transfer to PVDF membrane (0.45 μm) using wet transfer system (25 mM Tris, 192 mM glycine, 20% methanol)

• Transfer at 100V for 1 hour or 30V overnight at 4°C

• Verify transfer efficiency with reversible staining (Ponceau S)

Immunodetection:

• Block membrane with 5% non-fat dry milk in TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature

• Dilute At1g28600 antibody 1:1000 in blocking solution

• Incubate membrane with primary antibody overnight at 4°C with gentle rocking

• Wash membrane 3 × 10 minutes with TBST

• Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature

• Wash membrane 3 × 10 minutes with TBST

• Apply ECL substrate and detect signal using appropriate imaging system

Critical Optimization Parameters:

Essential Controls:

• Positive control: Wild-type Arabidopsis tissue known to express At1g28600

• Negative control: If available, include At1g28600 knockout/knockdown tissue

• Loading control: Detect a constitutively expressed protein (e.g., actin, tubulin, GAPDH) to normalize loading

This protocol should be optimized based on specific laboratory conditions and sample characteristics .

What sample preparation methods maximize At1g28600 detection sensitivity?

Effective detection of At1g28600 in plant tissues requires optimized sample preparation protocols that preserve protein integrity while minimizing interfering compounds. The following comprehensive approach maximizes detection sensitivity:

Tissue Selection and Harvesting:

• Identify tissues with optimal At1g28600 expression (if known)

• Harvest at appropriate developmental stage

• Collect samples at consistent times to account for potential circadian regulation

• Flash-freeze immediately in liquid nitrogen

• Store at -80°C until processing if immediate extraction is not possible

Extraction Buffer Optimization:
Different extraction buffers can significantly impact protein recovery and quality:

Critical Additives:

• Protease inhibitor cocktail (complete, EDTA-free) - essential for all extractions

• Phosphatase inhibitors (if phosphorylation status is important)

• PVPP (2%) to remove phenolic compounds in plant tissues

• DTT or β-mercaptoethanol as reducing agents

• EDTA (1-5 mM) to inhibit metalloproteases

Tissue Disruption Methods:

• Mortar and pestle with liquid nitrogen: Thorough but labor-intensive

• Bead mill homogenizer: Efficient for multiple samples

• Avoid excessive heat generation during homogenization

Protein Concentration and Clean-up:

• TCA/acetone precipitation: Removes interfering compounds and concentrates proteins

  • Add 4 volumes of ice-cold 10% TCA in acetone to sample

  • Incubate at -20°C for 1 hour to overnight

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

  • Wash pellet with ice-cold acetone

  • Resuspend in appropriate buffer

• Protein concentration determination:

  • Use Bradford or BCA assay compatible with your buffer components

  • Prepare standard curves in the same buffer as samples

Sample Storage Recommendations:

• Add 1× Laemmli buffer to prepared samples for Western blot

• Heat at 95°C for 5 minutes (adjust based on protein characteristics)

• Store at -20°C for short term or -80°C for long term

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

These optimized sample preparation methods will maximize the likelihood of successful At1g28600 detection while minimizing background and potential artifacts.

What controls are essential for At1g28600 antibody-based immunoprecipitation?

Immunoprecipitation (IP) experiments with At1g28600 antibody require rigorous controls to ensure specificity and reliability. The following comprehensive set of controls should be included:

Essential Controls for At1g28600 Immunoprecipitation:

• Input Sample Control:

  • Reserve 5-10% of pre-IP lysate

  • Run alongside IP samples in Western blot

  • Verifies presence of target protein in starting material

  • Allows calculation of IP efficiency

• Negative Control Antibody:

  • Perform parallel IP with non-specific IgG from same species (rabbit )

  • Process identically to experimental IP

  • Identifies non-specific binding to antibody or beads

  • Critical for distinguishing genuine interactions from background

• Genetic Controls:

  • Include samples from At1g28600 knockout/knockdown lines if available

  • Should show reduced/absent signal compared to wild-type

  • Provides strong evidence for antibody specificity

  • Essential for validating novel interactions

• Bead-Only Control:

  • Incubate lysate with beads without antibody

  • Identifies proteins binding non-specifically to matrix

  • Particularly important when studying new protein interactions

• Peptide Competition Control:

  • Pre-incubate antibody with excess immunizing peptide/protein

  • Compare with standard IP

  • Specific signals should be substantially reduced

Control Implementation Matrix:

Additional Advanced Controls:

• Reciprocal IP:

  • If studying interaction partners, perform reverse IP with antibody against the partner

  • Confirms interaction is detectable in both directions

  • Strengthens evidence for genuine interaction

• Stringency Gradient:

  • Perform parallel IPs with increasing salt or detergent concentrations

  • Helps distinguish strong specific interactions from weak non-specific binding

  • True interactors persist under higher stringency conditions

• Crosslinking Control:

  • If using crosslinking, include non-crosslinked samples

  • Distinguishes direct from indirect interactions

  • Helps identify transient interactions

By implementing these controls, researchers can confidently distinguish genuine At1g28600 interactions from experimental artifacts, ensuring robust and reproducible results in protein interaction studies .

What dilution ranges are recommended for At1g28600 antibody across applications?

Optimal dilution of At1g28600 antibody varies by application, sample type, and detection method. The following recommendations provide starting points for method optimization based on typical polyclonal antibody performance and the product specifications :

Recommended Dilution Ranges by Application:

*Applications requiring additional validation as they are not listed in the tested applications

Dilution Optimization Strategy:

• Initial Assessment:

  • Begin with the manufacturer's recommended dilution or the starting dilution above

  • Include positive control (wild-type Arabidopsis)

  • Include negative control (At1g28600 knockout if available or primary antibody omission)

• Titration Series:

  • Prepare 3-5 different dilutions spanning the recommended range

  • Keep all other experimental conditions constant

  • Example Western blot titration: 1:500, 1:1000, 1:2000, 1:5000

  • Evaluate signal-to-noise ratio at each dilution

• Optimization Parameters by Application:

For Western Blot:

• If signal is too strong: Increase antibody dilution or reduce protein loading

• If signal is too weak: Decrease antibody dilution, increase protein loading, or extend exposure time

• If background is high: Increase antibody dilution, optimize blocking, or increase washing stringency

For ELISA:

• Create a standard curve with known concentrations of recombinant protein

• Test multiple antibody dilutions to determine the linear range of detection

• Optimize blocking agent (BSA vs. milk) to minimize background

For Immunoprecipitation:

• Adjust antibody amount based on target protein abundance

• Optimal ratio typically 2-5 μg antibody per mg of total protein

• Include detergent optimization to reduce non-specific binding

• Application-Specific Considerations:

  • For Western blot: Detection method (ECL vs. fluorescent) affects optimal dilution

  • For ELISA: Plate type and coating conditions influence sensitivity

  • For microscopy: Fixation method significantly impacts epitope accessibility and optimal dilution

These recommendations should be considered starting points, with optimal conditions determined empirically for each specific experimental system and sample type .

What troubleshooting strategies address common issues with At1g28600 antibody experiments?

When working with At1g28600 antibody, researchers may encounter various technical challenges. The following troubleshooting guide addresses common issues and their solutions:

Western Blot Troubleshooting:

ELISA Troubleshooting:

Immunoprecipitation Troubleshooting:

Step-by-Step Optimization Process:

• Systematic Parameter Variation:

  • Change only one variable at a time

  • Document all modifications and results

  • Create a decision tree based on outcomes

• Signal Enhancement Strategies:

  • For weak signals: Enhance detection system (sensitive ECL substrate, signal amplification)

  • For low abundance proteins: Concentrate samples through immunoprecipitation

  • For masked epitopes: Test different extraction and denaturation conditions

• Specificity Confirmation:

  • Peptide competition assay

  • Testing in knockout/knockdown lines

  • Comparing multiple antibodies against the same target

• Application-Specific Optimization:

  • Western blot: Optimize transfer time and membrane type (PVDF vs. nitrocellulose)

  • ELISA: Test different plate types and coating buffers

  • IP: Compare different bead types (Protein A vs. G) and elution conditions

By implementing these troubleshooting strategies, researchers can overcome technical challenges and generate reliable, reproducible data with the At1g28600 antibody across various experimental applications .