At3g50940 Antibody

What is the AT3G50940 protein and why is it studied?

AT3G50940 encodes an AAA-ATPase protein in Arabidopsis thaliana that belongs to the AAA+ (ATPases Associated with diverse cellular Activities) superfamily. These proteins are involved in various cellular processes including protein degradation, membrane fusion, DNA replication, and microtubule dynamics. The study of AT3G50940 contributes to our understanding of fundamental cellular mechanisms in plants, particularly in energy-dependent processes. Research with AT3G50940 antibodies allows for detection, quantification, and localization of this protein in various experimental contexts, providing insights into its function and regulation within the plant cell.

How should AT3G50940 antibody be stored to maintain optimal activity?

The AT3G50940 antibody is typically provided in lyophilized form and requires specific storage conditions to preserve its functionality. Store the lyophilized antibody in a manual defrost freezer to prevent degradation. Avoid repeated freeze-thaw cycles as they can significantly reduce antibody activity and specificity through protein denaturation and aggregation. When the product is shipped at 4°C, store it immediately at the recommended temperature upon receipt . After reconstitution, aliquot the antibody into small volumes suitable for single experiments to minimize freeze-thaw cycles. Document each freeze-thaw cycle and validate antibody performance if multiple cycles occur, as sensitivity may decrease proportionally with each thawing event.

What validation methods should be used to confirm AT3G50940 antibody specificity?

The gold standard for antibody validation involves testing the antibody in paired wild-type and knockout (KO) cell lines. For plant antibodies like AT3G50940, this approach remains optimal but requires generating appropriate plant mutants. A comprehensive validation approach should include:

• Western blot analysis: Compare protein detection between wild-type and knockout/knockdown samples to confirm the antibody recognizes a band of the expected molecular weight that disappears in the absence of target protein .

• Immunoprecipitation testing: Verify the antibody can specifically pull down the target protein from complex lysates, confirmed by mass spectrometry or western blot analysis .

• Immunofluorescence assessment: Confirm proper subcellular localization pattern that is absent in knockout controls .

• Cross-reactivity testing: Evaluate potential cross-reactivity with closely related proteins, particularly important for plant research where gene families are common.

Recent large-scale antibody validation studies have shown that approximately 20-30% of protein studies use ineffective antibodies, underscoring the critical importance of robust validation before experimental use .

What are the recommended experimental controls when using the AT3G50940 antibody?

When designing experiments with the AT3G50940 antibody, implement the following controls to ensure reliable results:

Additionally, include concentration gradients of the antibody to determine optimal working dilutions for each application (Western blot, immunoprecipitation, or immunofluorescence) . These controls collectively provide confidence in experimental outcomes and help troubleshoot potential issues.

How can epitope accessibility issues be addressed when AT3G50940 antibody shows inconsistent results across different experimental techniques?

Epitope accessibility challenges with AT3G50940 antibody may manifest as discrepancies between techniques (e.g., successful Western blot but failed immunofluorescence). This discrepancy often stems from protein conformational differences across applications. To address this systematically:

• Fixation optimization: Test multiple fixation methods (paraformaldehyde, methanol, acetone) at different concentrations and durations. Aldehyde-based fixatives may mask epitopes by creating protein cross-links, while organic solvents might better preserve certain epitopes.

• Antigen retrieval: Implement heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) for formalin-fixed samples. For plant tissues specifically, enzymatic antigen retrieval using proteinase K or trypsin at optimized concentrations can expose hidden epitopes.

• Detergent permeabilization: Compare different detergents (Triton X-100, Tween-20, saponin) at varying concentrations (0.1-0.5%) to improve antibody access to intracellular targets without disrupting protein structure.

• Blocking optimization: Test alternative blocking agents (BSA, normal serum, commercial blocking buffers) to reduce background while preserving epitope accessibility.

• Antibody incubation conditions: Systematically vary temperature (4°C, room temperature, 37°C), duration (2h to overnight), and antibody concentration to identify optimal binding conditions .

What strategies can overcome cross-reactivity when AT3G50940 antibody detects multiple bands in Western blot analysis?

When encountering multiple bands in Western blot analysis using AT3G50940 antibody, implement this systematic troubleshooting approach:

• Bioinformatic analysis: Conduct sequence homology searches to identify proteins with similar epitopes to AT3G50940, particularly within the AAA-ATPase family. Predict potential cross-reactive proteins based on epitope sequence conservation.

• Gradient gel electrophoresis: Use 4-20% gradient gels to achieve better separation of proteins with similar molecular weights, allowing clearer distinction between specific and non-specific bands.

• Competitive blocking: Pre-incubate the antibody with excess purified AT3G50940 peptide corresponding to the immunogen. Specific bands should disappear while non-specific bands remain.

• Knockout validation: Compare Western blot patterns between wild-type and AT3G50940 knockout/knockdown samples. The specific band should disappear or be significantly reduced in knockout samples .

• Immunoprecipitation-mass spectrometry: Perform immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody, confirming whether multiple bands represent different forms of AT3G50940 or cross-reactive proteins.

• Alternative antibody comparison: Test multiple antibodies targeting different epitopes of AT3G50940 and compare banding patterns. Consistent bands across different antibodies increase confidence in specificity.

• Sample preparation optimization: Compare different lysis buffers, protease inhibitors, and protein denaturation conditions to determine if multiple bands result from degradation products or post-translational modifications.

Recent large-scale antibody validation studies have shown that only about two-thirds of protein targets have effective antibodies available, highlighting the importance of rigorous validation strategies .

How can AT3G50940 antibody be utilized for quantitative analysis of protein expression across different plant tissues or treatment conditions?

For quantitative analysis of AT3G50940 protein expression across different experimental conditions, implement this methodological approach:

• Sample preparation standardization: Establish a consistent protein extraction protocol with standardized tissue amounts, buffer composition, and homogenization methods. For plant tissues specifically, address extraction challenges by optimizing buffer pH (7.4-8.0) and detergent concentrations to handle varying cell wall compositions across tissue types.

• Protein normalization: Quantify total protein using BCA or Bradford assays before loading, and verify equal loading using housekeeping proteins appropriate for plant tissues (e.g., actin, tubulin, or GAPDH) that remain stable across your experimental conditions.

• Standard curve generation: Prepare a standard curve using recombinant AT3G50940 protein at known concentrations to establish a quantitative relationship between signal intensity and protein amount.

• Quantitative Western blot optimization:

  • Use fluorescent secondary antibodies rather than chemiluminescence for wider linear dynamic range

  • Determine optimal primary antibody concentration through titration experiments

  • Capture images before signal saturation occurs

  • Perform technical replicates (minimum three) and biological replicates (minimum three)

• Digital image analysis: Use specialized software (ImageJ, Image Studio, etc.) with consistent background subtraction methods and region-of-interest selection.

• Statistical analysis: Apply appropriate statistical tests based on experimental design, typically ANOVA with post-hoc tests for multiple condition comparisons, including error propagation calculations.

• Validation with orthogonal methods: Confirm quantitative changes using complementary approaches such as mass spectrometry or qRT-PCR (acknowledging the limitation that mRNA levels may not correlate with protein levels) .

What is the optimal protocol for using AT3G50940 antibody in Western blot applications?

The following optimized Western blot protocol has been developed specifically for AT3G50940 antibody based on validation studies with similar antibodies:

• Sample preparation:

  • Extract proteins using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and protease inhibitor cocktail

  • Sonicate briefly (3 × 10s pulses) to shear genomic DNA

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

  • Quantify protein concentration using Bradford assay

• Gel electrophoresis:

  • Load 20-40 μg protein per lane on a 10% SDS-PAGE gel

  • Include molecular weight markers and positive/negative controls

  • Run at 100V through stacking gel, then 150V through resolving gel

• Transfer:

  • Transfer proteins to PVDF membrane (0.45 μm pore size) using wet transfer system

  • Transfer at 100V for 60 minutes in cold transfer buffer containing 20% methanol

  • Verify transfer efficiency with reversible protein stain (Ponceau S)

• Blocking:

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

• Primary antibody incubation:

  • Dilute AT3G50940 antibody 1:1000 in 5% BSA in TBST

  • Incubate overnight at 4°C with gentle rocking

  • Expected molecular weight for AT3G50940: approximately 50-55 kDa

• Washing:

  • Wash membrane 3 × 10 minutes with TBST

• Secondary antibody incubation:

  • Use HRP-conjugated anti-rabbit or anti-mouse IgG (depending on primary antibody host) at 1:5000 dilution

  • Incubate for 1 hour at room temperature

• Detection:

  • Wash membrane 3 × 10 minutes with TBST

  • Apply ECL substrate and image using appropriate detection system

  • For quantitative analysis, use fluorescent secondary antibodies and appropriate imaging systems

This protocol should be optimized for each specific research application, with special attention to antibody dilution and incubation conditions.

How should AT3G50940 antibody be utilized for immunoprecipitation experiments?

The following validated immunoprecipitation protocol is recommended for AT3G50940 antibody applications:

• Cell/tissue lysis:

  • Homogenize plant tissue in IP lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease inhibitors)

  • Use a gentler lysis buffer (0.5% NP-40) if preserving protein complexes is critical

  • Clarify lysate by centrifugation at 14,000 × g for 15 minutes at 4°C

  • Pre-clear lysate with protein A/G beads for 1 hour at 4°C

• Antibody binding:

  • Use 2-5 μg of AT3G50940 antibody per 1 mg of total protein

  • Add antibody to pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

• Immunoprecipitation:

  • Add 30-50 μl protein A/G magnetic beads (pre-washed)

  • Incubate for 2-4 hours at 4°C with gentle rotation

  • Collect beads using magnetic rack

• Washing:

  • Wash beads 4-5 times with IP wash buffer (same as lysis buffer but with 0.1% detergent)

  • For the final wash, use buffer without detergent

• Elution:

  • For Western blot analysis: Add 30-50 μl of 1× SDS sample buffer and heat at 95°C for 5 minutes

  • For mass spectrometry: Use non-denaturing elution buffer (0.1 M glycine, pH 3.0)

• Controls to include:

  • Input sample (5% of pre-cleared lysate)

  • IgG control (same amount of non-specific IgG matching the host species of AT3G50940 antibody)

  • Beads-only control (no antibody added)

• Verification:

  • Analyze eluted samples by Western blot using a different AT3G50940 antibody (recognizing a different epitope) if available

  • For protein interaction studies, confirm results with reciprocal IP experiments

Recent antibody validation studies have shown that success in immunoprecipitation is less common than in Western blotting, making it especially important to optimize conditions specifically for this application .

What considerations are critical when using AT3G50940 antibody for immunofluorescence in plant tissues?

When implementing immunofluorescence with AT3G50940 antibody in plant tissues, address these critical aspects:

• Sample preparation:

  • For fixed tissue sections: Use 4% paraformaldehyde in PBS for 24 hours, then embed in paraffin or optimal cutting temperature compound

  • For protoplasts or cell suspensions: Fix with 4% paraformaldehyde for 15-30 minutes

  • Cut sections at 5-10 μm thickness for optimal antibody penetration

• Cell wall considerations:

  • Pre-treat sections with cell wall-degrading enzymes (cellulase, pectinase) to improve antibody penetration

  • Optimize enzymatic digestion time to balance antigen preservation with accessibility

• Permeabilization:

  • Use 0.1-0.3% Triton X-100 in PBS for 10-15 minutes

  • Alternatively, use methanol permeabilization (-20°C, 10 minutes) which may better preserve certain epitopes

• Blocking:

  • Block with 3-5% BSA or normal serum (matching secondary antibody host) with 0.1% Triton X-100 in PBS

  • Include 0.1-0.3% Tween-20 to reduce non-specific binding

  • Add 0.1% glycine to quench aldehyde groups from fixation

• Primary antibody incubation:

  • Dilute AT3G50940 antibody 1:100-1:500 in blocking buffer

  • Incubate overnight at 4°C in a humidified chamber

  • For thick sections, extend incubation time or perform at room temperature

• Controls:

  • Secondary antibody-only control

  • Peptide competition control (antibody pre-incubated with immunizing peptide)

  • Negative control tissue (AT3G50940 knockout or knockdown if available)

• Signal amplification options:

  • Consider tyramide signal amplification for low-abundance proteins

  • Use high-sensitivity detection systems (e.g., quantum dots or newer generation fluorophores)

• Counterstaining:

  • Include nuclear counterstain (DAPI or Hoechst)

  • Add cellular compartment markers to provide context for AT3G50940 localization

• Autofluorescence management:

  • Pre-treat sections with 0.1% sodium borohydride to reduce fixative-induced autofluorescence

  • Use spectral imaging and linear unmixing to separate antibody signal from plant autofluorescence

How can researchers troubleshoot weak or absent signal when using AT3G50940 antibody?

When encountering weak or absent signal with AT3G50940 antibody, implement this systematic troubleshooting approach:

• Antibody functionality verification:

  • Test antibody in a simple dot blot with recombinant AT3G50940 protein

  • Verify antibody hasn't degraded by comparing with a new lot or alternative antibody

• Protein expression validation:

  • Confirm target protein expression in your sample using RT-PCR

  • Use positive control samples known to express AT3G50940

• Sample preparation optimization:

  • For Western blot: Test different lysis buffers and protein extraction methods

  • For immunofluorescence: Try alternative fixation methods and antigen retrieval techniques

  • For immunoprecipitation: Adjust lysis conditions and bead types

• Protocol parameter adjustment:

  • Systematic titration of antibody concentration (try 2-5× higher concentration)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize secondary antibody dilution and incubation conditions

  • Test different detection methods (ECL substrates of varying sensitivity, fluorescent detection)

• Epitope accessibility enhancement:

  • For Western blot: Try both reducing and non-reducing conditions

  • For immunofluorescence: Implement heat-induced or enzymatic antigen retrieval

  • For fixed tissues: Test different permeabilization methods and durations

• Signal amplification strategies:

  • Use polymer-based detection systems

  • Implement tyramide signal amplification

  • Try biotin-streptavidin amplification systems

• Background reduction:

  • Optimize blocking conditions (concentration, buffer, time)

  • Include 0.1-0.3% Tween-20 in wash buffers

  • Perform more frequent and longer washing steps

Recent antibody validation studies have found that approximately one-third of commercially available antibodies fail to detect their target proteins reliably, highlighting the importance of thorough validation and troubleshooting protocols .

How can researchers differentiate between specific and non-specific signals when using AT3G50940 antibody?

Distinguishing specific from non-specific signals requires implementation of multiple complementary strategies:

• Knockout/knockdown validation:

  • Compare signals between wild-type samples and AT3G50940 knockout or RNAi knockdown samples

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

  • This represents the gold standard for specificity validation

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide (5-10× molar excess)

  • In parallel, incubate antibody with unrelated peptide

  • Specific signals should disappear only with the specific peptide competition

• Multiple antibody comparison:

  • Test at least two antibodies targeting different epitopes of AT3G50940

  • Signals detected by both antibodies are more likely to be specific

  • Discrepancies between antibodies require further investigation

• Molecular weight verification:

  • Compare observed molecular weight with predicted size

  • Account for post-translational modifications that alter migration

  • Verify with recombinant protein control

• Subcellular localization consistency:

  • Confirm localization pattern matches known or predicted localization

  • Use compartment-specific markers as co-staining controls

  • Inconsistent localization may indicate non-specific binding

• Signal intensity correlation:

  • Compare signal intensity with known expression levels across tissues/conditions

  • Verify that signal changes correlate with expected biological responses

  • Inconsistent correlation suggests non-specific binding

• Signal-to-noise ratio analysis:

  • Calculate signal-to-background ratios under different conditions

  • Specific signals typically have higher signal-to-noise ratios

  • Implement appropriate background subtraction methods

Recent large-scale antibody validation studies have emphasized that the gold standard for specificity validation requires comparison between wild-type and knockout samples, with other methods providing supporting but not definitive evidence .

What strategies enable reliable quantification of AT3G50940 protein across different experimental conditions?

For accurate quantification of AT3G50940 protein, implement this comprehensive approach:

• Rigorous experimental design:

  • Include biological replicates (minimum n=3) and technical replicates

  • Randomize sample processing order to prevent systematic bias

  • Include standard curves using recombinant AT3G50940 protein

• Sample preparation standardization:

  • Standardize cell/tissue collection, storage, and processing

  • Use identical protein extraction methods across all samples

  • Quantify total protein using consistent methods (BCA or Bradford assay)

  • Prepare all samples in a single batch when possible

• Loading control optimization:

  • For Western blot: Use multiple housekeeping proteins as loading controls

  • Verify loading control stability across experimental conditions

  • Consider total protein normalization methods (Ponceau S, Coomassie, SYPRO Ruby)

• Quantitative detection methods:

  • For Western blot: Use fluorescent secondary antibodies rather than chemiluminescence

  • For immunofluorescence: Implement standardized image acquisition settings

  • For ELISA: Include standard curves on each plate

• Linear dynamic range verification:

  • Test serial dilutions of samples to confirm measurements fall within linear range

  • Avoid signal saturation in all quantification methods

  • Document linear range for each specific experimental setup

• Image analysis standardization:

  • Use consistent region-of-interest selection methods

  • Apply identical background subtraction across all samples

  • Implement automated analysis workflows to reduce operator bias

• Statistical analysis rigor:

  • Test data for normality before selecting parametric/non-parametric tests

  • Apply appropriate statistical tests for experimental design

  • Report effect sizes alongside p-values

  • Consider power analysis to determine adequate sample size

Research on antibody validation has shown that rigorous quantification requires not only specific antibodies but also standardized protocols and proper statistical analysis, with particular attention to dynamic range limitations .