At1g30790 Antibody

What is At1g30790 Antibody and what does it target?

At1g30790 Antibody (CSB-PA874555XA01DOA) is a polyclonal antibody raised in rabbits that specifically targets the At1g30790 protein from Arabidopsis thaliana (Mouse-ear cress). This antibody is generated using recombinant Arabidopsis thaliana At1g30790 protein as the immunogen. The target protein (UniProt: Q9SY20) is involved in plant cellular processes, making this antibody a valuable tool for plant biology research .

What are the optimal storage conditions for At1g30790 Antibody?

The At1g30790 Antibody should be stored at -20°C or -80°C upon receipt. It's critical to avoid repeated freeze-thaw cycles as these can degrade antibody quality and reduce binding efficacy. For research requiring frequent access to the antibody, it's recommended to prepare small aliquots after initial reconstitution to minimize freeze-thaw degradation. This practice is consistent with standard protocols for preserving antibody functionality across multiple experiments .

How should At1g30790 Antibody be reconstituted before use?

For proper reconstitution of At1g30790 Antibody, add 20 μl of sterile water to each tube before use. After reconstitution, the antibody is delivered in liquid form containing a storage buffer composed of preservative (0.03% Proclin 300) and constituents (50% Glycerol, 0.01M PBS, pH 7.4). Always remember to briefly spin the tubes prior to opening to collect any material that might adhere to the cap or sides of the tube .

What applications has At1g30790 Antibody been validated for?

The At1g30790 Antibody has been specifically tested and validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications. These techniques allow for the detection and quantification of the target protein in complex biological samples. The antibody's affinity purification enhances its specificity for these applications, making it suitable for precise identification of the antigen in research settings .

What controls should be included when using At1g30790 Antibody in Western Blot experiments?

When designing Western Blot experiments with At1g30790 Antibody, include the following controls:

• Positive control: Sample known to express At1g30790 protein (e.g., wild-type Arabidopsis thaliana tissue)

• Negative control: Sample lacking At1g30790 expression (e.g., knockout mutant)

• Loading control: Detection of a housekeeping protein (like actin or tubulin) to verify equal loading

• Primary antibody control: Omitting primary antibody to identify non-specific binding of secondary antibody

• Blocking peptide control: Pre-incubating antibody with immunizing peptide to confirm specificity

This methodical approach aligns with rigorous experimental design practices seen in immunoblotting protocols for other plant antibodies .

How can researchers optimize Western Blot protocols specifically for At1g30790 Antibody?

For optimal Western Blot results with At1g30790 Antibody, consider the following methodology:

• Dilution: Start with the recommended 1:10,000 dilution, then adjust based on signal strength

• Blocking: Use 5% non-fat dry milk or BSA in TBST buffer (may need optimization)

• Incubation time: Primary antibody incubation overnight at 4°C often yields better results than shorter incubations

• Washing: Extensive washing (4-5 times for 5 minutes each) with TBST reduces background

• Detection method: Choose chemiluminescence for higher sensitivity or colorimetric methods for stable results

• Exposure time: For digital imaging systems, test multiple exposure times to find optimal signal-to-noise ratio

This systematic approach helps achieve clear, reproducible results while minimizing background and non-specific binding .

How does the polyclonal nature of At1g30790 Antibody affect experimental design and data interpretation?

The polyclonal nature of At1g30790 Antibody has significant implications for research design. Unlike monoclonal antibodies that recognize a single epitope, this polyclonal antibody contains a heterogeneous mixture of immunoglobulins that recognize multiple epitopes on the target protein. This characteristic offers advantages in detecting native proteins and proteins with post-translational modifications, as it increases the likelihood of target recognition.

What considerations should be made regarding potential cross-reactivity when using At1g30790 Antibody?

While At1g30790 Antibody is specifically designed for Arabidopsis thaliana, researchers working with related plant species should consider potential cross-reactivity. The antibody's polyclonal nature increases the likelihood of recognizing conserved epitopes across species. When working with non-Arabidopsis samples:

• Validate specificity through preliminary Western blots comparing Arabidopsis and your species of interest

• Perform sequence alignments between At1g30790 and homologous proteins in your species

• Include appropriate negative controls (e.g., pre-immune serum)

• Consider pre-absorbing the antibody with non-target proteins if cross-reactivity is observed

• Validate results using complementary techniques (e.g., mass spectrometry)

This methodical approach helps distinguish between specific binding and cross-reactivity in experimental systems .

How can researchers assess At1g30790 Antibody binding characteristics to enhance experimental reproducibility?

To characterize and optimize At1g30790 Antibody binding for improved reproducibility:

• Perform titration experiments to determine optimal antibody concentration for your specific application

• Characterize binding kinetics through techniques like surface plasmon resonance (SPR)

• Assess affinity constants to understand the strength of antibody-antigen interactions

• Determine specificity through competitive binding assays with purified recombinant At1g30790

• Evaluate potential Fc-mediated effects that might influence experimental outcomes

Understanding these binding characteristics allows for refined experimental protocols and facilitates more accurate interpretation of results, especially when quantitative analysis is required .

What are common issues when using At1g30790 Antibody in Western blot applications and how can they be resolved?

When troubleshooting Western blots with At1g30790 Antibody, consider these methodological solutions for common problems:

This systematic approach helps isolate and address specific issues in the experimental workflow .

How can researchers distinguish between true At1g30790 signal and potential artifacts?

Distinguishing genuine At1g30790 signal from artifacts requires a multi-faceted validation approach:

• Molecular weight verification: Compare observed band to the expected molecular weight of At1g30790

• Knockout/knockdown validation: Compare wild-type samples with those where At1g30790 expression is reduced or eliminated

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to block specific binding

• Multiple detection methods: Confirm findings using alternative techniques (e.g., immunoprecipitation, mass spectrometry)

• Signal correlation analysis: Verify if signal intensity correlates with expected biological changes (e.g., stress response)

This comprehensive validation strategy strengthens confidence in experimental findings and reduces the risk of misinterpreting artifacts as genuine signal .

What approaches can resolve contradictory data when using At1g30790 Antibody across different experimental conditions?

When faced with contradictory results across experiments using At1g30790 Antibody, implement this structured troubleshooting methodology:

• Standardize protein extraction: Use identical buffer systems and extraction protocols across experiments

• Control for post-translational modifications: Consider phosphorylation, ubiquitination, or other modifications that might affect antibody recognition

• Normalize environmental conditions: Control for variables like plant age, growth conditions, and stress exposure

• Consider temporal dynamics: At1g30790 expression may vary with developmental stage or diurnal rhythms

• Evaluate technical variability: Statistical analysis of technical replicates can distinguish biological variance from technical noise

• Perform interlaboratory validation: Collaborate with other researchers to verify results using identical protocols

This systematic approach helps identify sources of variability and reconcile apparently contradictory findings .

How can At1g30790 Antibody be used in combination with other antibodies for co-localization studies?

For co-localization studies examining At1g30790 in relation to other proteins, implement this methodological approach:

• Selection of compatible secondary antibodies: Choose secondary antibodies with distinct fluorophores that have minimal spectral overlap

• Sequential immunostaining protocol:

  • Fix and permeabilize tissue samples appropriately for plant cellular structures

  • Block with 5% normal serum corresponding to secondary antibody host

  • Incubate with At1g30790 Antibody at 1:500-1:2,000 dilution

  • Apply first secondary antibody and wash thoroughly

  • Block again before applying second primary antibody

  • Apply second secondary antibody with distinct fluorophore

• Controls for co-localization:

  • Single antibody controls to establish baseline signal and bleed-through

  • Non-specific IgG controls to assess background

  • Absorption controls with recombinant proteins

This approach enables precise spatial determination of At1g30790 relative to other cellular components .

What considerations should be made when designing immunoprecipitation experiments with At1g30790 Antibody?

For successful immunoprecipitation of At1g30790 and its interaction partners:

• Buffer optimization: Start with standard IP buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% NP-40) and adjust detergent concentration based on protein solubility

• Antibody coupling: Consider covalently coupling At1g30790 Antibody to protein A/G beads to prevent antibody co-elution

• Pre-clearing lysates: Incubate plant extracts with protein A/G beads alone to reduce non-specific binding

• Cross-linking considerations:

  • For transient interactions, consider mild cross-linking with DSP or formaldehyde

  • Optimize cross-linking time and concentration to preserve interactions without obscuring epitopes

• Elution strategies: Compare different elution methods (pH, competitive peptide, SDS) to maximize recovery without antibody contamination

• Validation of interactions: Confirm pulled-down proteins with reciprocal IP or alternative techniques like proximity ligation assay

This methodical approach maximizes the chances of capturing genuine protein interactions while minimizing artifacts .