BIA1 Antibody

What is BIA1 and what are its key characteristics?

BIA1 (also known as TRIM11) in mammals is a member of the TRIM/RBCC protein family that functions in innate immune responses and transcriptional regulation. The human version has a canonical amino acid length of 468 residues and a protein mass of 52.8 kilodaltons, with three identified isoforms. It is primarily localized in the nucleus and cytoplasm of cells and is widely expressed across many tissue types . In contrast, in plant biology, BIA1 (BRASSINOSTEROID INACTIVATOR1/ABNORMAL SHOOT1) is a BAHD acyltransferase that regulates brassinosteroid homeostasis through acetylation of castasterone .

What are the main applications for BIA1 antibodies in research?

BIA1 antibodies are primarily used in three key applications:

• Western Blot (WB) - For detection and quantification of BIA1 protein in cell or tissue lysates

• Enzyme-Linked Immunosorbent Assay (ELISA) - For quantitative measurement of BIA1 in solution

• Immunohistochemistry (IHC) - For visualization of BIA1 localization in tissue sections

For plant-specific BIA1 antibodies, Western Blot and ELISA are the common validated applications, particularly for research with Arabidopsis .

How do I select the appropriate BIA1 antibody for my research?

When selecting a BIA1 antibody, consider these critical factors:

For TRIM11/BIA1 research in mammalian systems, antibodies with human and rodent reactivity are available. For plant science, specific antibodies for Arabidopsis BIA1 exist .

How should I optimize Western blot protocols for BIA1/TRIM11 detection?

For optimal detection of BIA1/TRIM11 via Western blot:

• Sample preparation:

  • Use lysis buffers containing protease inhibitors

  • For nuclear-cytoplasmic proteins like BIA1, consider subcellular fractionation

  • Load 20-50 μg total protein per lane

• Gel separation and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution around the 53 kDa range

  • Transfer to PVDF or nitrocellulose membranes (60-90 minutes at 100V)

• Antibody incubation:

  • Block with 5% non-fat milk or 3-5% BSA in TBST (1 hour, room temperature)

  • Dilute primary antibody 1:500-1:2000 (optimize based on specific antibody)

  • Incubate overnight at 4°C for best results

  • Use appropriate HRP-conjugated secondary antibody (typically 1:5000-1:10000)

• Detection controls:

  • Expected band size: ~53 kDa for human BIA1/TRIM11

  • Include positive control samples from tissues known to express BIA1

What are the critical parameters for immunohistochemical detection of BIA1?

For successful immunohistochemical detection of BIA1:

• Tissue preparation:

  • Fixation: 10% neutral buffered formalin (24-48 hours)

  • Antigen retrieval: Heat-induced epitope retrieval (citrate buffer pH 6.0 or EDTA pH 9.0)

  • Section thickness: 4-5 μm for optimal antibody penetration

• Antibody protocol:

  • Blocking: 1-2 hours with serum matching secondary antibody species

  • Primary antibody: Optimize dilution (typically 1:100-1:500)

  • Incubation time: Overnight at 4°C for maximum sensitivity

  • Detection system: HRP/DAB or fluorescent secondary antibodies

• Essential controls:

  • Positive control: Tissue with known BIA1 expression

  • Negative control: Primary antibody omission

  • Specificity control: Pre-absorption with immunizing peptide

  • Compare staining pattern to known nuclear and cytoplasmic localization

How can I employ BIA1 antibodies in an ELISA system?

For developing an ELISA system using BIA1 antibodies:

• Assay formats:

  • Direct ELISA: Immobilize sample, detect with labeled anti-BIA1

  • Sandwich ELISA: Capture with one anti-BIA1 antibody, detect with another

  • Competitive ELISA: Compete sample BIA1 with standard for antibody binding

• Optimization parameters:

  • Coating concentration: 1-10 μg/ml antibody for capture

  • Blocking agent: 1-5% BSA or casein to reduce background

  • Sample dilution: Determine through standard curve analysis

  • Antibody concentration: Titrate to determine optimal signal-to-noise ratio

  • Incubation times: 1-2 hours at room temperature or overnight at 4°C

• Validation approach:

  • Spike-and-recovery experiments to assess matrix effects

  • Parallelism testing to ensure linearity across dilutions

  • Include standards of known concentration for quantification

  • Assess inter- and intra-assay variability (<15% CV)

How can I use BIA1 antibodies to study protein-protein interactions?

Several techniques utilizing BIA1 antibodies can elucidate protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate BIA1 using validated antibodies and analyze co-precipitating proteins

  • Use gentle lysis conditions to preserve protein-protein interactions

  • Include appropriate controls (IgG control, reverse Co-IP)

  • Western blot to detect specific interaction partners

• Proximity Ligation Assay (PLA):

  • Allows visualization of interactions (<40 nm) between BIA1 and potential partners

  • Requires primary antibodies from different species against each protein

  • Provides in situ detection of interactions with spatial resolution

  • Particularly useful for BIA1 given its dual nuclear-cytoplasmic localization

• Biacore SPR analysis:

  • Surface plasmon resonance can characterize binding kinetics

  • Immobilize antibody to capture BIA1, then flow potential binding partners

  • Alternatively, immobilize BIA1 and test antibody binding characteristics4

How can I distinguish between different BIA1/TRIM11 isoforms?

To differentiate between the three reported isoforms of human BIA1/TRIM11:

• Isoform-specific detection:

  • Select antibodies targeting unique epitopes in specific isoforms

  • Use high-resolution SDS-PAGE (gradient gels) to separate closely-sized isoforms

  • Complement with RT-PCR using isoform-specific primers

• Analytical approach:

  • First identify which isoforms your antibody can detect (check with manufacturer)

  • Run protein standards of known isoforms alongside your samples

  • For definitive identification, consider mass spectrometry following immunoprecipitation

• Experimental validation:

  • Express recombinant isoforms as positive controls

  • Use siRNA targeting specific isoforms to confirm band identity

  • Consider 2D gel electrophoresis for improved separation

What approaches can be used to study BIA1 in plant brassinosteroid signaling?

For plant scientists studying BIA1/ABS1 in brassinosteroid regulation:

• Enzymatic activity analysis:

  • In vitro acylation assays using purified recombinant BIA1

  • Use acetyl-CoA as a donor (rather than myristoyl-CoA)

  • Monitor acylation of castasterone (CS) or 24-epiCS

  • Analyze products via thin-layer chromatography (TLC)

• Genetic approaches:

  • Analyze brassinosteroid-related phenotypes in bia1 mutants

  • The dominant bia1-1D and bia1-2D mutants show severe BR-deficient phenotypes

  • Loss-of-function bia1-3 shows BR overaccumulation phenotypes

  • Rescue experiments with exogenous brassinosteroids

• Biochemical characterization:

  • Express BIA1 as GST-tagged protein for enzymatic studies

  • Monitor BR catabolism through acetylation

  • Analyze BR metabolites via chromatographic techniques

What are common challenges in detecting BIA1 and how can they be addressed?

Common challenges and solutions for BIA1 detection:

• Weak or absent signal in Western blot:

  • Ensure complete extraction of nuclear proteins (BIA1 is nuclear-cytoplasmic)

  • Increase protein loading (40-60 μg)

  • Try different antibody concentrations and incubation times

  • Use more sensitive detection systems (ECL prime or femto)

  • Verify expression level in your specific cell type/tissue

• Non-specific bands:

  • Increase washing stringency (more washes, higher salt)

  • Optimize blocking conditions (try BSA instead of milk)

  • Consider monoclonal antibodies for higher specificity

  • Validate with BIA1 knockdown/knockout controls

• High background in immunohistochemistry:

  • Optimize blocking (both serum and protein blocking)

  • Increase washing duration and frequency

  • Titrate primary antibody to minimize non-specific binding

  • Use biotin blocking if employing avidin-biotin systems

How should I validate the specificity of BIA1 antibodies?

A comprehensive validation approach includes:

• Genetic validation:

  • Test antibody in BIA1/TRIM11 knockout or knockdown samples

  • Compare with wild-type or overexpression systems

  • Expected result: Signal should diminish with KD/KO and increase with overexpression

• Peptide competition:

  • Pre-incubate antibody with the immunizing peptide

  • Run parallel assays with blocked and unblocked antibody

  • Specific signal should disappear or significantly decrease

• Orthogonal validation:

  • Compare results from multiple antibodies to different BIA1 epitopes

  • Correlate protein detection with mRNA expression

  • For plant BIA1, confirm enzyme activity with purified protein

• Cross-reactivity testing:

  • Test against samples from species with different sequence homology

  • Check for expected patterns across multiple applications (WB, IHC, ELISA)

How can I quantitatively analyze BIA1 expression data?

For quantitative analysis of BIA1 expression:

• Western blot densitometry:

  • Use integrated density values rather than peak intensity

  • Normalize to appropriate loading controls (β-actin, GAPDH)

  • Employ analysis software such as ImageJ/FIJI

  • Report data as fold-change relative to control

• Immunohistochemistry quantification:

  • Scoring methods: H-score (combines intensity and percentage positive cells)

  • Digital image analysis using appropriate thresholding

  • Blind analysis by multiple observers for unbiased assessment

  • Compare nuclear vs. cytoplasmic staining separately

• Statistical considerations:

  • Minimum sample size: n=3 independent biological replicates

  • Statistical tests: t-test for two conditions, ANOVA for multiple conditions

  • Account for multiple testing when analyzing across conditions

  • Report exact p-values and confidence intervals