At3g05950 Antibody

What is the At3g05950 protein and why is it scientifically significant?

At3g05950, also known as Germin-like protein subfamily 1 member 7 (GLP17), is a plant protein with a putative role in defense mechanisms in Arabidopsis thaliana. While it shares structural similarities with oxalate oxidases, current evidence suggests it likely lacks this enzymatic activity despite conservation of the active site. The protein is found in the secreted extracellular space (apoplast) and is classified within the Germin protein family. Its significance lies in understanding plant immune responses and stress adaptation mechanisms, making antibodies against this target valuable tools for investigating these biological processes.

What forms of At3g05950 antibodies are commercially available for research?

Research-grade At3g05950 antibodies are typically available as polyclonal antibodies raised in rabbits, with the immunogen being a KLH-conjugated peptide selected from specific regions of the Arabidopsis thaliana At3g05950 protein sequence. These antibodies are often supplied in lyophilized format as immunogen-affinity purified serum in PBS pH 7.4, requiring reconstitution before use . Some suppliers may offer custom antibody production services with lead times of approximately 14-16 weeks for made-to-order antibodies targeting specific epitopes of the At3g05950 protein.

How should At3g05950 antibodies be stored to maintain optimal activity?

For maximum stability and activity retention, store lyophilized At3g05950 antibodies at -20°C until ready for use. Upon reconstitution with the recommended volume of sterile water (typically 50 μl for a 50 μg quantity), the antibody should continue to be stored at -20°C . To prevent activity loss from repeated freeze-thaw cycles, it is advisable to prepare small aliquots of the reconstituted antibody. Before opening, briefly centrifuge tubes to collect any material that may have adhered to the cap or sides during storage or shipping . For some antibody preparations, an equal volume of glycerol (ACS grade or better) can be added to the reconstituted antibody for a final concentration of 50% glycerol, which may extend storage stability at -20°C .

What controls are essential when designing experiments with At3g05950 antibody?

When designing experiments with At3g05950 antibody, four types of controls are essential to ensure specificity and validity of results:

• Unstained cells/tissue control: This addresses autofluorescence issues that may create false positives, particularly important when using fluorescent detection methods.

• Negative control samples: Use tissue or cell populations known not to express At3g05950 to verify antibody specificity. For plant experiments, this could include tissues from knockout mutants or species known not to express the protein (such as Brassica napus, Populus sp., Rosa chinensis, or Triticum aestivum, which have been reported as non-reactive with some At3g05950 antibodies) .

• Isotype control: Include an antibody of the same class as your primary antibody but directed against an irrelevant antigen to assess non-specific binding, particularly binding through Fc receptors.

• Secondary antibody-only control: This is crucial for indirect detection methods to identify background signal from non-specific secondary antibody binding .
  These controls help distinguish true signal from background and validate the specificity of antigen-antibody interactions in your experimental system.

What are optimal blocking conditions for Western blot applications of At3g05950 antibody?

For Western blot applications using At3g05950 antibody, effective blocking is crucial to minimize background and enhance signal-to-noise ratio. Based on published protocols, recommended blocking conditions include:

• Use 2% blocking reagent (such as GE Healthcare blocking reagent) in TBS-T buffer.

• Block membranes for 1 hour at room temperature with gentle agitation.

• For alternative blocking, consider 10% normal serum from the same host species as the labeled secondary antibody (important: do NOT use serum from the same species as the primary antibody).

• For plant protein samples, additional blocking agents may be necessary to reduce plant-specific background .
  After blocking, incubate with the primary At3g05950 antibody at a recommended dilution of 1:1000 for 1 hour at room temperature with agitation, followed by thorough washing steps (one 15-minute and three 5-minute washes) in TBS-T buffer .

How should protein samples be prepared to optimize At3g05950 detection?

For optimal detection of At3g05950 protein in plant samples, the following sample preparation protocol is recommended:

• Extraction buffer composition: Use 50 mM Tris-HCl pH 7.5, 10% glycerol, 150 mM NaCl, 0.1% NP-40, 1 mM PMSF, and 1× protease inhibitor cocktail (such as from Roche).

• Sample quantity: Load approximately 40 μg of total protein per lane for standard detection methods.

• Electrophoresis conditions: Separate proteins on a 4-20% gradient SDS-PAGE gel to accommodate the expected molecular weight of At3g05950 (approximately 64 kDa).

• Transfer parameters: Transfer to PVDF membrane for approximately 1 hour using standard transfer conditions.

• Pre-treatment considerations: For young seedlings (e.g., 5-day-old dark-grown Arabidopsis), gentle extraction methods are recommended to preserve protein integrity .
  This preparation method has been demonstrated to effectively isolate and maintain the integrity of At3g05950 protein for subsequent antibody detection.

What is the recommended protocol for Western blot detection of At3g05950?

The following optimized Western blot protocol is recommended for specific detection of At3g05950:
Sample Preparation and Separation:

• Extract total protein using buffer containing 50 mM Tris-HCl pH 7.5, 10% glycerol, 150 mM NaCl, 0.1% NP-40, 1 mM PMSF, and 1× protease inhibitor cocktail.

• Load 40 μg of total protein per lane.

• Separate on 4-20% gradient SDS-PAGE.

• Transfer to PVDF membrane for 1 hour using standard transfer buffer.
  Immunodetection:

• Block membrane with 2% blocking reagent in TBS-T for 1 hour at room temperature with agitation.

• Incubate with primary At3g05950 antibody diluted 1:1000 in blocking buffer for 1 hour at room temperature with agitation.

• Wash membrane: rinse briefly twice, then wash once for 15 minutes and three times for 5 minutes in TBS-T.

• Incubate with HRP-conjugated secondary antibody (anti-rabbit IgG) diluted 1:10,000 for 1 hour at room temperature with agitation.

• Wash as in step 3.

• Develop using chemiluminescence detection reagent for approximately 5 minutes .
  Expected Results:
  The At3g05950 protein should appear at approximately 64 kDa, though variations may occur based on post-translational modifications or sample preparation conditions.

How can I optimize antibody dilutions for maximum sensitivity and specificity?

To optimize antibody dilutions for At3g05950 detection, perform a systematic titration experiment following these guidelines:

• Primary antibody optimization:

  • Start with the recommended dilution of 1:1000 for Western blotting

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

  • Use identical sample loadings and consistent detection conditions

  • Evaluate both signal intensity and background levels

• Secondary antibody optimization:

  • Begin with manufacturer's recommended dilution (typically 1:10,000 for HRP-conjugated anti-rabbit IgG)

  • Test dilution range (e.g., 1:5000, 1:10,000, 1:20,000)

  • Select the dilution providing optimal signal-to-noise ratio

• Incubation parameters:

  • Test different incubation times (1 hour vs. overnight at 4°C for primary antibody)

  • Compare different incubation temperatures (room temperature vs. 4°C)

  • Determine if gentle agitation improves antibody binding uniformity
    The optimal dilution will produce the strongest specific signal with minimal background. Document all optimization parameters for experimental reproducibility and consistent results across experiments .

What techniques are available for measuring At3g05950 antibody binding affinity?

Several techniques can be employed to assess the binding affinity of At3g05950 antibodies to their target antigen:

• Surface Plasmon Resonance (SPR) Spectroscopy:

  • Provides real-time measurement of binding kinetics

  • Determines association (kon) and dissociation (koff) rate constants

  • Calculates equilibrium dissociation constant (KD)

  • Requires specialized equipment such as Biacore systems

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Indirect ELISA with varying antibody concentrations

  • Calculate apparent KD from binding curves

  • More accessible than SPR but provides less detailed kinetic information

• Homogeneous Mobility Shift Assays:

  • Measures the formation of antibody-antigen complexes

  • Can determine relative binding affinities

  • Useful for comparing different antibody lots or preparations

• Antigen Binding Tests (ABTs):

  • Various formats including pH-shift-anti-idiotype antigen binding tests

  • Can incorporate electrochemiluminescence (ECL) detection for increased sensitivity

  • Allows detection of antibody-antigen interactions at low concentrations
    The choice of method depends on available equipment, required precision, and experimental goals. For most research applications, ELISA-based methods provide a practical approach to assessing antibody affinity.

How can I detect low-abundance At3g05950 protein in complex plant samples?

Detecting low-abundance At3g05950 protein in complex plant samples requires specialized approaches to enhance sensitivity:

• Sample Enrichment Strategies:

  • Perform subcellular fractionation to isolate the apoplast/extracellular fraction where At3g05950 is localized

  • Use immunoprecipitation with At3g05950 antibody to concentrate the target protein before analysis

  • Consider protein extraction methods optimized for secreted proteins

• Enhanced Detection Methods:

  • Implement amplified detection systems such as tyramide signal amplification (TSA)

  • Utilize more sensitive chemiluminescent substrates (e.g., femto-level sensitivity reagents)

  • Consider switching to fluorescent-based Western detection with specialized scanners

• Alternative Detection Platforms:

  • Consider electrochemiluminescence (ECL) binding assays which can detect antigens at concentrations down to 5-64 μg/L

  • Explore capillary electrophoresis-based immunoassays for higher sensitivity

  • Investigate the potential of liquid chromatography-mass spectrometry approaches for highly specific detection

• Protocol Optimization:

  • Extend primary antibody incubation time to overnight at 4°C

  • Increase antibody concentration while carefully controlling for specificity

  • Use low-protein binding tubes and filters to prevent sample loss during preparation
    These approaches can be combined as needed, with appropriate controls to ensure that the enhanced signal represents true target detection rather than artifacts or background.

What are common causes of non-specific binding with At3g05950 antibody and how can they be addressed?

Non-specific binding when using At3g05950 antibody can arise from several sources. The following table outlines common causes and corresponding solutions:

How can specificity of At3g05950 antibody be validated in different plant species?

Validating the specificity of At3g05950 antibody across different plant species requires a systematic approach:

How should I quantify and normalize Western blot data for At3g05950 expression analysis?

For rigorous quantitative analysis of At3g05950 expression by Western blot, follow these methodological steps:

• Image Acquisition:

  • Capture images using a digital imaging system with linear dynamic range

  • Avoid saturated pixels that will prevent accurate quantification

  • Collect multiple exposure times to ensure working within the linear range

• Band Intensity Measurement:

  • Use image analysis software (ImageJ, Image Studio, etc.)

  • Define consistent measurement areas for all bands and backgrounds

  • Subtract local background from each band measurement

• Normalization Approaches:

  • Loading Control Method: Normalize to housekeeping proteins (e.g., actin, GAPDH, tubulin)

  • Total Protein Normalization: Use stain-free gels or total protein stains (Ponceau S, SYPRO Ruby)

  • Multiple Reference Gene Approach: Use the geometric mean of several housekeeping proteins for more robust normalization

• Calculation Methods:

  • Calculate relative expression as: (Target protein intensity / Normalization factor)

  • For treatment comparisons, express as fold-change relative to control conditions

  • For time course experiments, consider area under the curve (AUC) measurements

• Statistical Analysis:

  • Perform experiments with at least three biological replicates

  • Apply appropriate statistical tests (t-test, ANOVA) based on experimental design

  • Report means, standard deviations/errors, and p-values
    This quantification approach enables reliable comparison of At3g05950 expression across different experimental conditions, treatments, or genotypes while controlling for technical variation.

What factors might affect the apparent molecular weight of At3g05950 in Western blots?

Several factors can influence the apparent molecular weight of At3g05950 in Western blot analysis, potentially causing deviation from the expected 64 kDa size:

• Post-translational Modifications:

  • Glycosylation may increase apparent molecular weight

  • Phosphorylation can cause band shifts of several kDa

  • Ubiquitination or SUMOylation would significantly increase molecular weight

• Sample Preparation Effects:

  • Incomplete denaturation may result in compact protein migration

  • Over-reduction can break internal disulfide bonds altering migration

  • Protein degradation during extraction creates lower MW bands

• Gel System Variables:

  • Acrylamide percentage affects migration patterns

  • Buffer composition influences protein-SDS interaction

  • Running conditions (voltage, temperature) impact migration

• Protein-Specific Properties:

  • Highly acidic or basic proteins may bind SDS irregularly

  • Proline-rich regions can cause anomalous migration

  • Intrinsically disordered regions affect SDS binding

• Experimental Artifacts:

  • Air bubbles in the gel create migration inconsistencies

  • Uneven heating during running causes "smile effect"

  • Edge effects from gel position
    When encountering unexpected molecular weights, confirm protein identity through additional methods such as immunoprecipitation followed by mass spectrometry, or expression validation in knockout and overexpression lines to ensure the observed band truly represents At3g05950.

How can contradictory results between antibody-based detection methods be reconciled?

When facing contradictory results between different antibody-based detection methods for At3g05950, a systematic reconciliation approach should be implemented:

• Method-Specific Considerations:

  Detection Method	Potential Limitations	Validation Approach
  Western Blot	Denatures proteins; epitopes may be altered	Compare native vs. reducing conditions
  ELISA	May detect denatured or fragmented proteins	Test with purified recombinant protein
  Immunohistochemistry	Fixation may mask epitopes	Compare multiple fixation methods
  Flow Cytometry	Surface accessibility issues	Validate with permeabilization controls

• Antibody Characterization:

  • Identify the exact epitope recognized by the antibody

  • Determine if epitope is accessible in all experimental conditions

  • Test multiple antibodies targeting different regions of At3g05950

• Biological Variables:

  • Assess if contradictions relate to different developmental stages

  • Determine if environmental conditions affect protein conformation

  • Consider tissue-specific post-translational modifications

• Experimental Reconciliation:

  • Design experiments that directly compare methods on identical samples

  • Include appropriate positive and negative controls for each method

  • Implement peptide competition assays to verify specificity

  • Use genetic approaches (knockout/overexpression) for definitive validation

• Molecular Resolution:

  • Consider mass spectrometry to definitively identify the protein

  • Implement proximity ligation assays to confirm spatial localization

  • Use alternative approaches like RNA-seq to correlate with protein expression
    By systematically evaluating method-specific limitations and implementing complementary validation approaches, seemingly contradictory results can often be reconciled to provide a more complete understanding of At3g05950 expression and function .