At5g55070 Antibody

What is the At5g55070 gene and its encoded protein?

At5g55070 is a gene ID from Arabidopsis thaliana that encodes one of the E2 subunits of the 2-oxoglutarate dehydrogenase complex (E2-OGDH1). This protein plays a crucial role in the tricarboxylic acid (TCA) cycle, participating in the conversion of 2-oxoglutarate to succinyl-CoA. The protein has the UniProt ID Q8H107-1 and functions in plant metabolism within mitochondria . When designing experiments involving this protein, it's important to consider its subcellular localization and enzymatic function.

What are the primary applications for At5g55070 antibodies?

At5g55070 antibodies are primarily used in plant research for detecting and studying the E2-OGDH1 protein. Common applications include western blotting, immunoprecipitation, immunohistochemistry, and ELISA. These antibodies are particularly valuable in studies investigating plant metabolism, mitochondrial function, and stress responses. The specificity of these antibodies allows researchers to monitor protein expression levels, protein-protein interactions, and post-translational modifications of E2-OGDH1 . When selecting an application, researchers should consider the specific experimental question and the nature of the biological sample.

How can I validate the specificity of At5g55070 antibodies?

Validating antibody specificity is crucial for obtaining reliable results. For At5g55070 antibodies, several approaches are recommended:

• Use positive controls (plant tissues known to express E2-OGDH1) and negative controls (tissues from knockout mutants or species lacking the protein)

• Perform pre-absorption tests with the immunizing peptide

• Compare results with alternative antibodies targeting the same protein

• Verify size and pattern by western blot against predicted molecular weight

• If possible, utilize mass spectrometry to confirm protein identity after immunoprecipitation

Researchers should document all validation steps and include these in their methodology sections when publishing results.

What is the expected cross-reactivity of At5g55070 antibodies with related proteins?

At5g55070 (E2-OGDH1) has a related isoform, E2-OGDH2 (encoded by AT4G26910), with which antibodies might cross-react depending on the epitope targeted. The antibody listed in the Agrisera database (Anti-E2-OGDH1/E2-OGDH2) is designed to recognize both isoforms . If studying only one isoform is crucial for your research, additional validation and potentially epitope-specific antibodies may be required. Cross-reactivity tests with recombinant proteins can help determine the exact specificity profile of the antibody.

How should I optimize protein extraction protocols for At5g55070 detection?

For optimal detection of E2-OGDH1 protein, consider these methodological steps:

• Start with fresh plant material and maintain cold conditions throughout extraction

• Use a mitochondria-enriched fraction to enhance detection sensitivity, as E2-OGDH1 is primarily localized in mitochondria

• Include protease inhibitors to prevent degradation

• Add reducing agents (like DTT or β-mercaptoethanol) to preserve protein structure

• Optimize buffer pH (typically 7.5-8.0) to maintain protein stability

• Consider gentle detergents (0.1-0.5% Triton X-100) for membrane solubilization

• Validate extraction efficiency with known mitochondrial markers

For challenging tissues, a comparative analysis of different extraction methods may be necessary to determine the optimal protocol.

What are the key considerations for designing competitive experiments with At5g55070 antibodies?

When designing competitive binding experiments:

• Use purified recombinant E2-OGDH1 protein or synthesized peptides corresponding to the antibody epitope

• Establish a dose-response curve with varying concentrations of the competitor

• Include appropriate controls (non-competing proteins/peptides)

• Pre-incubate the antibody with the competitor before adding to the sample

• Consider the binding kinetics and allow adequate incubation time

• Maintain consistent temperature and buffer conditions across experiments

• Quantify binding inhibition using sensitive detection methods

The results should be analyzed using appropriate statistical methods to determine IC50 values or other relevant parameters.

How can I distinguish between E2-OGDH1 (At5g55070) and E2-OGDH2 (At4g26910) in experimental settings?

Distinguishing between these related proteins requires careful experimental design:

• Employ antibodies targeting unique epitopes specific to each isoform, if available

• Utilize genetic approaches with knockout/knockdown lines for each gene

• Perform RNA interference or CRISPR-based gene editing to selectively reduce expression

• Use mass spectrometry to identify isoform-specific peptides

• Conduct immunoprecipitation followed by mass spectrometry

• Analyze expression patterns in tissues with known differential expression of these isoforms

• Consider using tagged versions of the proteins in transgenic lines

Remember that the antibody Anti-E2-OGDH1/E2-OGDH2 from Agrisera is designed to recognize both isoforms, so additional approaches may be necessary for isoform-specific studies.

What are the optimal western blotting conditions for At5g55070 antibodies?

For optimal western blotting results with At5g55070 antibodies:

• Sample preparation:

  • Use fresh plant material and efficient extraction buffers

  • Include appropriate protease inhibitors

  • Denature samples at 70-95°C for 5-10 minutes

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal separation

  • Load 10-30 μg of total protein per lane (adjust based on expression level)

• Transfer conditions:

  • Semi-dry or wet transfer at 15-20V overnight at 4°C for better transfer efficiency

  • Use PVDF membranes for stronger protein binding

• Blocking and antibody incubation:

  • Block with 3-5% non-fat dry milk or BSA in TBST

  • Dilute primary antibody 1:1000 to 1:5000 (optimize empirically)

  • Incubate overnight at 4°C with gentle agitation

  • Use secondary antibody at 1:5000 to 1:10000 dilution

• Detection and analysis:

  • Use ECL substrates appropriate for your expected signal intensity

  • Consider testing different exposure times

How should I approach troubleshooting weak or absent signals with At5g55070 antibodies?

When facing weak or absent signals, consider this methodical troubleshooting approach:

• Protein extraction issues:

  • Verify extraction efficiency with control antibodies for abundant proteins

  • Ensure protein integrity by checking with Ponceau S staining

  • Consider enriching for mitochondrial fractions

• Antibody-related issues:

  • Test different antibody concentrations (perform a titration)

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

  • Verify antibody quality with a dot blot of purified antigen

  • Check antibody storage conditions and expiration

• Detection system problems:

  • Use more sensitive detection reagents

  • Extend exposure time

  • Ensure secondary antibody compatibility

  • Check for interference from blocking agents

• Biological considerations:

  • Confirm protein expression under your experimental conditions

  • Consider if treatments might affect protein stability or expression

  • Test positive control samples known to express the protein

Document all troubleshooting steps systematically to identify the most likely cause of the problem.

What controls should be included in immunoprecipitation experiments with At5g55070 antibodies?

A robust immunoprecipitation experiment with At5g55070 antibodies should include these controls:

• Input control: Sample before immunoprecipitation to assess starting material

• No-antibody control: Beads without antibody to identify non-specific binding

• Isotype control: Unrelated antibody of the same isotype to assess specificity

• Pre-immune serum control: If available, to establish baseline reactivity

• Knockout/knockdown control: Material from plants lacking or with reduced E2-OGDH1

• Competition control: Pre-incubation with immunizing peptide/protein

• Reverse IP: Using antibodies against suspected interacting partners

• Denaturing vs. native conditions: To distinguish direct vs. indirect interactions

These controls help validate genuine interactions and distinguish them from experimental artifacts, enhancing data reliability and interpretability.

How should I quantify and normalize At5g55070 protein levels in comparative studies?

For reliable quantification and normalization of E2-OGDH1 protein levels:

• Quantification approaches:

  • Use digitized densitometry of western blot bands

  • Apply fluorescence-based quantification for wider dynamic range

  • Consider multiple technical replicates (3-4) per biological sample

• Normalization strategies:

  • Normalize to total protein (measured by Ponceau S or Coomassie staining)

  • Use established housekeeping proteins (caution: verify stability under your conditions)

  • Consider mitochondrial markers (e.g., VDAC) for normalization when focusing on mitochondrial function

  • Apply multiple normalization references for robust analysis

• Statistical analysis:

  • Perform appropriate statistical tests based on experimental design

  • Consider biological (not just technical) replicates

  • Report both raw and normalized data when possible

• Validation:

  • Corroborate protein level changes with transcript analysis

  • Verify biological significance through functional assays

What are the considerations when analyzing At5g55070 expression under stress conditions?

When analyzing E2-OGDH1 expression under stress conditions:

• Temporal dynamics:

  • Include multiple time points to capture expression kinetics

  • Consider both early and late responses to stress

• Stress-specific considerations:

  • For oxidative stress: monitor ROS levels alongside protein expression

  • For nutrient stress: measure relevant metabolite levels

  • For temperature stress: consider protein stability changes

  • For pathogen stress: monitor pathogen progression

• Post-translational modifications:

  • Assess potential changes in protein phosphorylation, acetylation, or other modifications

  • Consider using phosphorylation-specific antibodies if relevant

• Protein activity vs. abundance:

  • Complement expression data with enzymatic activity assays

  • Consider the relationship between protein levels and functional outcomes

• Data integration:

  • Correlate protein expression with metabolomic changes in the TCA cycle

  • Analyze mitochondrial function alongside protein expression

How can I interpret conflicting results when studying At5g55070 protein interactions?

When faced with conflicting results in protein interaction studies:

• Methodological differences:

  • Compare detection methods (co-IP vs. Y2H vs. BiFC vs. FRET)

  • Assess buffer conditions that might affect interactions

  • Consider native vs. denatured conditions

  • Evaluate tag positions that might interfere with binding sites

• Biological considerations:

  • Investigate tissue-specific or developmental stage-specific differences

  • Consider post-translational modifications affecting interactions

  • Evaluate environmental conditions influencing protein associations

  • Assess the role of accessory proteins in complex formation

• Experimental validation:

  • Use multiple complementary techniques to verify interactions

  • Perform domain mapping to identify interaction regions

  • Consider directed mutagenesis of predicted interaction sites

  • Use structural biology approaches when feasible

• Reconciliation strategies:

  • Develop integrated models that explain context-dependent interactions

  • Consider kinetic aspects of protein associations

  • Evaluate concentration-dependent effects on complex formation

How does the At5g55070 antibody compare to other plant metabolism antibodies?

The following table summarizes key characteristics of the At5g55070 (E2-OGDH) antibody compared to other metabolism-related antibodies:

This table helps researchers select appropriate antibodies for comparative metabolism studies and design multiplexed experiments for investigating metabolic enzyme complexes.