At3g05150 Antibody

What is the At3g05150 antibody and what protein does it target?

The At3g05150 antibody targets the protein encoded by the At3g05150 gene in Arabidopsis thaliana. This antibody is typically developed as a polyclonal or monoclonal preparation that recognizes specific epitopes on this plant protein. When selecting an At3g05150 antibody for research purposes, it's critical to verify whether it has been raised against the whole protein or specific peptide sequences. Most commonly, these antibodies are produced in goat, rabbit, or mouse hosts and may be available as affinity-isolated antibody preparations in buffered aqueous glycerol solutions for optimal stability .

What are the optimal storage conditions for At3g05150 antibodies?

To maintain antibody functionality and prevent degradation, At3g05150 antibodies should be stored at 2-8°C for short-term use (up to one month) and at -20°C for long-term storage. Avoid repeated freeze-thaw cycles as these can dramatically reduce antibody efficacy through protein denaturation. Most commercial preparations come in glycerol-containing buffers (typically 50% glycerol) with preservatives like sodium azide (approximately 2.15 mM) to prevent microbial growth and maintain stability . When handling the antibody, always use sterile techniques and store aliquots in non-frost-free freezers to prevent temperature fluctuations.

How should I validate the specificity of my At3g05150 antibody?

Antibody validation requires multiple complementary approaches:

• Western blot analysis: Run protein extracts from wild-type and At3g05150 knockout/knockdown plants to confirm the antibody detects a band of the expected molecular weight only in wild-type samples.

• Immunoprecipitation: Perform IP experiments followed by mass spectrometry to identify all proteins pulled down by the antibody.

• Cross-reactivity testing: Test reactivity against related plant species to determine specificity.

• Blocking peptide experiments: Pre-incubate the antibody with the immunizing peptide before application to verify that specific binding is eliminated.

• Signal localization: Compare immunofluorescence patterns with established subcellular localization data for the At3g05150 protein.

These validation steps are essential to prevent data misinterpretation due to antibody polyreactivity or polyspecificity, which has been observed in many therapeutic and research antibodies .

What are the recommended dilutions for At3g05150 antibody in different applications?

These dilutions should be optimized for each specific antibody lot and experimental system. For initial experiments, perform a dilution series to determine the optimal signal-to-noise ratio for your specific application. When using enzyme-conjugated secondary antibodies, similar optimization approaches should be employed - for example, alkaline phosphatase-conjugated secondary antibodies are typically used at 1:5,000-1:10,000 dilutions for Western blot applications .

How can I optimize protein extraction protocols for detecting At3g05150 in plant tissues?

For optimal At3g05150 protein extraction from plant tissues:

• Tissue selection: Choose appropriate tissues where At3g05150 is expressed, based on transcriptomic data.

• Extraction buffer optimization:

  • Start with a standard buffer (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100)

  • Add protease inhibitors (complete protease inhibitor cocktail)

  • Include reducing agents (5 mM DTT) if the protein contains disulfide bonds

  • For membrane-associated proteins, use stronger detergents (0.5% SDS or 1% sodium deoxycholate)

• Mechanical disruption: Use liquid nitrogen grinding followed by brief sonication to ensure complete cell lysis while maintaining protein integrity.

• Post-extraction processing:

  • Centrifuge at 12,000 × g for 10 minutes at 4°C

  • Collect supernatant and quantify protein concentration

  • Add SDS-PAGE loading buffer and heat at 95°C for 5 minutes (or 70°C for 10 minutes if protein is heat-sensitive)

This methodology improves extraction efficiency while minimizing protein degradation, which is particularly important for plant proteins that may have unique post-translational modifications or structural characteristics .

What controls should I include when using At3g05150 antibody for immunoblotting?

A robust immunoblotting experiment with At3g05150 antibody requires several controls:

• Positive control: Protein extract from tissues known to express At3g05150 or recombinant At3g05150 protein

• Negative controls:

  • Protein extract from At3g05150 knockout/knockdown plants

  • Pre-immune serum (for polyclonal antibodies)

  • Isotype control (for monoclonal antibodies)

  • Secondary antibody-only control to detect non-specific binding

• Loading control: Probing for a housekeeping protein (e.g., actin, tubulin, or GAPDH) to normalize protein loading

• Competition control: Pre-incubation of the antibody with excess immunizing peptide to verify signal specificity

• Cross-reactivity assessment: Testing against protein extracts from related species or paralogs to evaluate specificity

Why might I observe multiple bands when using At3g05150 antibody in Western blots?

Multiple bands in Western blots using At3g05150 antibody could result from several factors:

• Post-translational modifications: The At3g05150 protein may undergo phosphorylation, glycosylation, or other modifications that alter its migration pattern.

• Alternative splicing: The At3g05150 gene may produce multiple isoforms with different molecular weights.

• Protein degradation: Incomplete protease inhibition during sample preparation can lead to degradation products.

• Cross-reactivity: The antibody may recognize epitopes present in related proteins.

• Non-specific binding: Particularly common with polyclonal antibodies or at low dilutions.

To address these issues:

• Optimize protein extraction with complete protease inhibitor cocktails

• Use freshly prepared samples

• Increase antibody dilution to reduce non-specific binding

• Perform peptide competition assays to confirm specificity

• Validate with knockout/knockdown samples

• Consider using different antibody clones that target distinct epitopes

Understanding the nature of additional bands requires careful validation experiments to distinguish between true isoforms and technical artifacts .

How can I reduce background signal when using At3g05150 antibody for immunofluorescence?

High background in immunofluorescence with At3g05150 antibody can be addressed through several optimization strategies:

• Sample fixation optimization:

  • Test different fixatives (4% paraformaldehyde, methanol, or acetone)

  • Optimize fixation time (typically 10-20 minutes)

  • Ensure complete permeabilization with appropriate detergents (0.1-0.5% Triton X-100)

• Blocking improvements:

  • Increase blocking agent concentration (5-10% normal serum from secondary antibody species)

  • Try alternative blocking agents (BSA, casein, or commercial blocking buffers)

  • Extend blocking time to 1-2 hours at room temperature

• Antibody dilution optimization:

  • Test serial dilutions to find optimal concentration

  • Incubate primary antibody longer at lower concentrations (overnight at 4°C)

  • Add 0.1% Tween-20 to antibody diluent to reduce non-specific binding

• Washing protocol enhancement:

  • Increase number of washes (5-6 times)

  • Extend wash durations (10 minutes each)

  • Use gentle agitation during washes

• Autofluorescence reduction:

  • Pretreat samples with 0.1% sodium borohydride

  • Include 0.1-1% Sudan Black B in mounting medium to quench plant tissue autofluorescence

These approaches target different aspects of background generation and can be combined for optimal results in plant tissue immunofluorescence applications .

What could cause loss of At3g05150 antibody reactivity over time?

Decreased antibody reactivity over time typically results from:

• Protein denaturation: Repeated freeze-thaw cycles disrupt antibody structure

• Microbial contamination: Compromised sterility leads to protein degradation

• Chemical degradation: Oxidation or hydrolysis of amino acid residues, particularly at elevated temperatures

• Aggregation: Formation of antibody aggregates reduces effective concentration

• Buffer issues: pH shifts or buffer component precipitation

Preventive measures include:

• Storing as small aliquots to minimize freeze-thaw cycles

• Adding sterile glycerol (final concentration 30-50%) for cryoprotection

• Maintaining strict sterile technique during handling

• Adding preservatives like sodium azide (0.02%) to prevent microbial growth

• Storing at recommended temperatures (-20°C for long-term)

• Monitoring storage conditions to prevent temperature fluctuations

If activity decreases, consider testing antibody concentration by absorbance at 280nm and performing a titration experiment to determine if higher concentrations restore function .

How can I use At3g05150 antibody for chromatin immunoprecipitation (ChIP) experiments?

For successful ChIP experiments with At3g05150 antibody:

• Crosslinking optimization:

  • Test different formaldehyde concentrations (1-3%) and incubation times (10-20 minutes)

  • Consider dual crosslinking with both formaldehyde and protein-specific crosslinkers for protein-DNA interactions

• Chromatin fragmentation:

  • Optimize sonication conditions to generate fragments of 200-500 bp

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Immunoprecipitation strategy:

  • Pre-clear chromatin with protein A/G beads to reduce background

  • Use 2-5 μg antibody per ChIP reaction

  • Include IgG control and input samples for normalization

  • Incubate overnight at 4°C with rotation

• Washing stringency:

  • Perform sequential washes with increasing stringency buffers

  • Monitor wash stringency to balance background reduction with signal preservation

• Elution and reversal of crosslinks:

  • Elute complexes at 65°C in SDS-containing buffer

  • Reverse crosslinks overnight at 65°C

  • Treat with RNase A and Proteinase K

• Analysis approaches:

  • Analyze by qPCR for known targets

  • Perform ChIP-seq for genome-wide binding profiling

This approach is particularly useful for determining if the At3g05150 protein interacts with chromatin, identifying DNA binding sites, and understanding its role in transcriptional regulation .

Can At3g05150 antibody be used to study protein-protein interactions through co-immunoprecipitation?

Co-immunoprecipitation (Co-IP) with At3g05150 antibody requires careful optimization:

• Lysis buffer selection:

  • Use gentle, non-denaturing buffers (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40)

  • Add protease and phosphatase inhibitors to preserve interactions

  • Consider including protein stabilizers like 5% glycerol

• Pre-clearing optimization:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Include appropriate blocking proteins (BSA or non-immune serum)

• Antibody immobilization methods:

  • Direct addition of antibody to lysate followed by protein A/G beads

  • Pre-coupling antibody to beads using chemical crosslinkers for improved specificity

  • Consider using commercially available immunoprecipitation kits optimized for plant proteins

• Washing stringency balance:

  • Adjust salt concentration (150-500 mM) to maintain specific interactions

  • Optimize detergent concentration to reduce background

  • Perform 4-6 washes with progressively reduced detergent concentration

• Elution strategies:

  • Gentle elution with excess immunizing peptide

  • pH elution (glycine buffer pH 2.8)

  • SDS elution for complete recovery

• Analysis methods:

  • Western blot for known interacting partners

  • Mass spectrometry for unbiased identification of interaction network

Co-IP can reveal physiologically relevant protein interactions that help elucidate the functional role of At3g05150 in plant cellular processes .

How can I quantitatively measure At3g05150 protein expression levels across different plant tissues?

To quantitatively assess At3g05150 protein expression across tissues:

• Sample preparation standardization:

  • Develop consistent extraction protocols for different tissue types

  • Normalize protein content using Bradford or BCA assays

  • Prepare all samples simultaneously to minimize batch effects

• Quantitative Western blot approach:

  • Include recombinant At3g05150 protein standard curve

  • Use fluorescent secondary antibodies for broader linear range

  • Capture images using systems with wide dynamic range (e.g., LI-COR Odyssey)

  • Normalize to multiple housekeeping proteins appropriate for the tissues being compared

• ELISA methodology:

  • Develop sandwich ELISA with capture and detection antibodies

  • Generate standard curves using recombinant protein

  • Process all samples in the same assay to minimize inter-assay variation

• Multiplexed protein analysis:

  • Consider automated capillary immunoassay systems (e.g., Wes, Jess)

  • Develop multiplexed assays to simultaneously quantify At3g05150 and normalizers

• Mass spectrometry-based quantification:

  • Employ absolute quantification using synthetic peptide standards

  • Consider label-free or isotope-labeled quantification approaches

  • Monitor multiple unique peptides from At3g05150 protein

This comprehensive approach enables reliable quantification across diverse tissue types with appropriate normalization strategies .

How do I interpret contradictory results between At3g05150 antibody-based methods and transcript analysis?

Discrepancies between protein detection and transcript levels are common and may reflect important biological phenomena:

• Post-transcriptional regulation:

  • mRNA stability differences affect the correlation between transcript and protein levels

  • miRNA-mediated repression may reduce protein without affecting transcript

  • Alternative splicing may produce transcripts that the antibody cannot detect

• Post-translational regulation:

  • Protein degradation rates influence steady-state protein levels

  • Modifications may mask epitopes recognized by the antibody

  • Protein localization changes may affect extraction efficiency

• Technical considerations:

  • Antibody may recognize specific protein conformations or modifications

  • Extraction protocols may not efficiently isolate certain protein populations

  • Sensitivity differences between transcript and protein detection methods

To resolve these discrepancies:

• Perform time-course experiments to identify temporal relationships

• Use multiple antibodies targeting different epitopes

• Employ protein synthesis/degradation inhibitors to assess protein turnover

• Consider proteasome inhibitor treatments to evaluate degradation contribution

• Examine subcellular fractions separately

• Compare results with complementary approaches like GFP-fusion proteins

Understanding these discrepancies often reveals important regulatory mechanisms controlling protein abundance independently of transcription .

How does antibody polyreactivity affect the interpretation of At3g05150 antibody results?

Antibody polyreactivity can significantly impact experimental interpretation:

• Sources of polyreactivity:

  • Conformational flexibility in antibody paratopes allowing diverse target binding

  • Charge-based interactions, particularly with highly charged proteins

  • Hydrophobic interactions, especially at higher antibody concentrations

  • Cross-reactivity with structurally homologous proteins

• Impact on experimental outcomes:

  • False positive signals in immunoassays

  • Misleading localization in imaging studies

  • Erroneous identification of interaction partners

  • Batch-to-batch variability in antibody performance

• Assessment strategies:

  • Test antibody binding to unrelated proteins (BSA, KLH, etc.)

  • Perform competitive binding assays

  • Evaluate binding under different salt/pH conditions

  • Compare results from multiple antibody sources/clones

  • Validate key findings with orthogonal methods

• Risk mitigation approaches:

  • Use more stringent washing conditions

  • Increase blocking stringency

  • Pre-adsorb antibodies with potential cross-reactants

  • Optimize antibody concentration to minimize non-specific binding

  • Validate with genetic controls (knockout/knockdown)

Recent studies have found that antibody polyreactivity is more common than previously recognized and represents a significant challenge for result interpretation, especially in complex biological systems like plant tissues .

What recent advances in experimental methodologies could improve At3g05150 protein research?

Emerging technologies offering advantages for At3g05150 protein research include:

• Proximity labeling approaches:

  • BioID or TurboID fusion proteins to identify physiological interaction partners

  • APEX2 for spatially resolved protein interaction mapping

  • Implementation allows in vivo identification of transient interactions

• Single-molecule imaging:

  • Super-resolution microscopy (PALM, STORM) for detailed localization studies

  • Single-particle tracking to analyze protein dynamics

  • Enables visualization of protein behavior in living plant cells

• Advanced proteomics:

  • Targeted proteomics (PRM/MRM) for absolute quantification without antibodies

  • Crosslinking mass spectrometry (XL-MS) for structural interaction studies

  • Thermal proteome profiling to assess protein stability and interactions

• CRISPR-based approaches:

  • Endogenous tagging for visualizing native protein

  • CUT&Tag for improved chromatin interaction mapping over traditional ChIP

  • Base editing for introducing specific mutations to study structure-function relationships

• Nanobody technology:

  • Development of plant-specific nanobodies for improved specificity

  • Intrabodies for tracking and manipulating proteins in living cells

  • Reduced background compared to conventional antibodies

These methodologies can complement or, in some cases, replace traditional antibody-based approaches, offering higher specificity, better spatial/temporal resolution, and more quantitative results for plant protein research .