zgc:153454 Antibody

What is zgc:153454 and what cellular function does it serve in zebrafish?

zgc:153454 (now known as med13b) encodes the Mediator of RNA polymerase II transcription subunit 13-like protein in zebrafish. This protein is part of the mediator complex that serves as a transcriptional coactivator for DNA-binding factors that regulate transcription by RNA polymerase II . The gene is located on chromosome 15 and has multiple available alleles with various mutations including nonsense and essential splice site variants .

The protein has 2102 amino acids based on the transcript ENSDART00000110267, containing 30 exons . As part of the mediator complex, it plays a critical role in regulating gene expression during development and in response to various stimuli. The importance of this gene is highlighted by the fact that the Zebrafish Mutation Project (ZMP) maintains multiple alleles of this gene for research purposes .

What are the key specifications of commercially available zgc:153454 antibodies?

Commercial zgc:153454 antibodies typically have the following specifications:

The antibody is typically shipped on blue ice and available in a 10mg size . When selecting an antibody, researchers should verify these specifications match their experimental requirements, particularly regarding species reactivity and validated applications.

What primary validation methods should be used for zgc:153454 antibody?

Validating a zgc:153454 antibody requires a multi-step approach to ensure specificity and reproducibility:

• Genetic validation: The most rigorous approach is to use genetic knockdown/knockout samples as negative controls. Given that multiple alleles (sa22623, sa22622, sa35864, etc.) are available for shipment through zebrafish mutation repositories, these can serve as excellent negative controls .

• Orthogonal validation: Compare protein expression results from antibody-based methods with antibody-independent methods such as mass spectrometry or mRNA quantification .

• Multiple antibody validation: Use different antibodies targeting different epitopes of zgc:153454 to confirm consistent results .

• Recombinant expression validation: Overexpress zgc:153454 in a model system and confirm increased signal .

• Immunoprecipitation followed by mass spectrometry: Confirm that the antibody specifically captures the target protein .

For each application (Western blot, ELISA, etc.), separate validation experiments should be conducted, as specificity in one application does not guarantee specificity in another . Document all validation experiments thoroughly, including positive and negative controls, for inclusion in publications .

What controls should be included when using zgc:153454 antibody in experimental protocols?

Proper experimental design with zgc:153454 antibody requires several controls:

• Positive control: Include samples known to express zgc:153454, such as wild-type zebrafish tissue. The recombinant immunogen protein provided with the antibody (200μg) can serve as a positive control .

• Negative control:

  • Use pre-immune serum (1ml included with antibody) as a primary antibody control

  • Include zgc:153454 knockout/knockdown samples if available (alleles sa22623, sa22622, etc. from mutation repositories)

  • Use tissues known not to express the target

• Loading controls: For Western blots, include housekeeping proteins (GAPDH, β-actin) to normalize expression levels .

• Secondary antibody only control: Omit primary antibody to detect potential non-specific binding of secondary antibody.

• Technical replicates: Perform at least three independent replicates for statistical validity.

• Batch controls: When comparing between experiments, include samples from previous batches to account for batch-to-batch variability .

The experimental design should also include proper optimization of antibody concentration. Using too much antibody can yield nonspecific results, while too little can lead to false-negative results. Determining signal-to-noise ratio and dynamic range is critical for quantitative experiments .

How should researchers optimize Western blotting protocols for zgc:153454 antibody?

Optimizing Western blotting with zgc:153454 antibody requires methodical adjustment of multiple parameters:

• Sample preparation:

  • Use fresh tissue samples or properly stored frozen samples (-80°C)

  • Include protease inhibitors during extraction

  • Determine optimal protein amount (typically 20-50μg total protein)

• Antibody dilution optimization:

  • Start with manufacturer's recommended dilution (typically 1:1000-1:5000)

  • Prepare a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

  • Evaluate signal-to-noise ratio at each dilution

• Blocking optimization:

  • Test different blocking agents (5% BSA vs. 5% non-fat milk)

  • Optimize blocking time (1-2 hours at room temperature or overnight at 4°C)

• Incubation conditions:

  • Compare different incubation times and temperatures

  • Test overnight incubation at 4°C vs. 1-2 hours at room temperature

• Washing stringency:

  • Optimize wash buffer composition (TBST or PBST with 0.05% to 0.1% Tween-20)

  • Adjust washing time and number of washes

• Detection system selection:

  • Choose appropriate secondary antibody (HRP-conjugated anti-rabbit IgG)

  • Select detection method (ECL, fluorescent, etc.) based on expected abundance

Expected molecular weight of zgc:153454 is approximately 230-240 kDa, but post-translational modifications may alter migration pattern . Document each optimization step systematically for reproducibility and include all relevant protocols in publications .

What approach is recommended for immunohistochemistry using zgc:153454 antibody?

For successful immunohistochemistry (IHC) with zgc:153454 antibody, follow this methodological approach:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde (PFA) for 24 hours

  • Process and embed in paraffin or prepare frozen sections

  • For zebrafish embryos/larvae, consider whole-mount immunostaining

• Antigen retrieval optimization:

  • Test multiple methods as this is critical for zgc:153454 detection

  • Compare heat-induced epitope retrieval methods:

    • Citrate buffer (pH 6.0)

    • EDTA buffer (pH 8.0)

    • Tris-EDTA buffer (pH 9.0)

  • Optimize retrieval time (10-30 minutes)

• Blocking and permeabilization:

  • Block with 5-10% normal serum from the same species as secondary antibody

  • Include 0.1-0.3% Triton X-100 for membrane permeabilization

  • Consider adding 1% BSA to reduce background

• Antibody incubation:

  • Dilute primary antibody appropriately (start with 1:100-1:500)

  • Incubate overnight at 4°C in humidified chamber

  • For fluorescent detection, protect from light during secondary antibody incubation

• Controls:

  • Include tissue sections from zgc:153454 knockout models

  • Use pre-immune serum in place of primary antibody

  • Include competing peptide control if available

• Counterstaining and mounting:

  • Use DAPI for nuclear counterstaining

  • Select appropriate mounting medium based on detection method

Remember that optimization needs to be performed for each specific tissue type. Document all optimization steps and include representative images of controls in publications .

How can researchers use zgc:153454 antibody to study protein-protein interactions?

Studying protein-protein interactions involving zgc:153454 requires specialized approaches:

• Co-immunoprecipitation (Co-IP):

  • Lyse zebrafish tissues or cells in non-denaturing buffer

  • Pre-clear lysates with Protein A/G beads

  • Immunoprecipitate with zgc:153454 antibody (2-5μg per mg of protein)

  • Analyze precipitates by Western blot for potential interaction partners

  • Include IgG control to identify non-specific interactions

• Proximity Ligation Assay (PLA):

  • Use zgc:153454 antibody with antibodies against suspected interaction partners

  • PLA signals appear only when proteins are in close proximity (<40nm)

  • Quantify interaction events using fluorescent microscopy

• Chromatin Immunoprecipitation (ChIP):

  • As a component of the mediator complex, zgc:153454 may be involved in DNA binding

  • Optimize crosslinking conditions (1% formaldehyde for 10-15 minutes)

  • Fragment chromatin to 200-500bp

  • Immunoprecipitate with 3-5μg zgc:153454 antibody

  • Analyze by qPCR or sequencing

• Immunofluorescence co-localization:

  • Perform double immunofluorescence with zgc:153454 and suspected partners

  • Analyze using confocal microscopy

  • Calculate co-localization coefficients (Pearson's, Mander's)

• Bimolecular Fluorescence Complementation (BiFC):

  • For validation of direct interactions identified in other assays

  • Clone zgc:153454 and partner proteins fused to split fluorescent protein fragments

  • Co-express in zebrafish cells and observe reconstituted fluorescence

Each approach provides different information about the interaction (direct/indirect, temporal, spatial), so combining multiple methods increases confidence in results. Include appropriate controls for each technique and validate key findings using orthogonal approaches .

How can zgc:153454 antibody be used to study developmental processes in zebrafish?

Using zgc:153454 antibody to study developmental processes requires temporal and spatial analysis:

• Developmental expression profiling:

  • Collect embryos at multiple developmental stages (e.g., 4-cell, blastula, gastrula, somitogenesis, 24hpf, 48hpf, 72hpf)

  • Process for either whole-mount immunostaining or protein extraction

  • Compare zgc:153454 expression levels and localization across stages

  • Correlate with known developmental events

• Tissue-specific expression analysis:

  • Perform immunohistochemistry on tissue sections from different developmental stages

  • Map expression patterns to specific cell types and tissues

  • Create expression atlas across development

• Functional studies:

  • Compare wild-type expression with mutant lines (available alleles: sa22623, sa22622, sa35864, etc.)

  • Correlate protein expression changes with developmental phenotypes

  • Use morpholino knockdown followed by rescue experiments to confirm specificity

• Co-expression studies:

  • Combine zgc:153454 antibody with markers for specific cell lineages

  • Determine co-expression patterns during development

  • Identify potential regulatory relationships

• Live imaging:

  • For advanced studies, consider using Fab fragments of zgc:153454 antibody

  • Label with fluorescent dyes for live imaging in transparent embryos

  • Track protein dynamics during developmental processes

This approach allows for comprehensive characterization of zgc:153454's role in zebrafish development. When reporting results, include clear timeline references and standardized staging nomenclature. Combine antibody-based detection with mRNA expression analysis for a more complete picture .

What strategies can overcome epitope masking when zgc:153454 protein interacts with other components?

Epitope masking can occur when zgc:153454 protein forms complexes or undergoes conformational changes. Several strategies can help overcome this challenge:

• Alternative extraction conditions:

  • Test different lysis buffers with varying detergent strengths

  • Compare native vs. denaturing conditions

  • Try sequential extraction protocols to disrupt different types of interactions

• Cross-linking followed by reversal:

  • Use reversible cross-linkers to stabilize complexes

  • Perform immunoprecipitation under cross-linked conditions

  • Reverse cross-links before immunoblotting

• Epitope retrieval optimization:

  • For fixed tissues, compare different antigen retrieval methods:

    • Heat-induced epitope retrieval at varying pH values

    • Enzymatic digestion (proteinase K, trypsin)

    • Detergent treatment during retrieval

• Multiple antibody approach:

  • Use different antibodies targeting distinct epitopes

  • Compare results to identify regions frequently masked by interactions

• Competitive binding assays:

  • Introduce excess recombinant immunogen peptide in controlled amounts

  • Monitor displacement of natural binding partners

• Denaturation strategies before antibody application:

  • For Western blots, ensure complete denaturation with SDS and reducing agents

  • For fixed tissues, try harsher retrieval conditions

Document all optimization attempts and include controls that demonstrate successful detection of the target protein. When reporting results, clearly state which extraction and detection conditions were ultimately successful .

How can researchers address batch-to-batch variability with zgc:153454 antibody?

Batch-to-batch variability is a common challenge with polyclonal antibodies like zgc:153454. Implement these methodological approaches to address this issue:

• Standardized validation protocol:

  • Develop a standard operating procedure (SOP) for validating each new batch

  • Include positive controls with known expression levels

  • Compare new batches against reference samples used with previous batches

  • Document sensitivity and specificity for each batch

• Bulk purchasing strategy:

  • Purchase multiple vials from the same lot when a reliable batch is identified

  • Store properly aliquoted at -80°C to maintain long-term stability

  • Reserve one vial for comparison testing with future batches

• Calibration curve approach:

  • For quantitative applications, generate a standard curve using recombinant protein

  • Normalize results across batches using these calibration curves

  • Report normalized values rather than raw intensities

• Bridging study design:

  • When changing batches mid-study, analyze a subset of samples with both batches

  • Develop a mathematical correction factor if needed

  • Include overlapping samples in all experimental runs

• Alternative validation methods:

  • Confirm key findings with orthogonal methods that don't depend on antibodies

  • Use knockout/knockdown controls with each new batch

Document lot numbers in all experimental records and publications. This approach not only addresses variability but also improves experimental reproducibility .

What are the most common causes of false positive and false negative results with zgc:153454 antibody?

Understanding potential sources of error is critical for accurate interpretation of results with zgc:153454 antibody:

Common causes of false positives:

• Cross-reactivity issues:

  • Antibody may recognize proteins with similar epitopes

  • Particularly relevant when using in species other than zebrafish

  • Confirm specificity through knockout controls and immunoprecipitation-mass spectrometry

• Excessive antibody concentration:

  • Too high concentration increases non-specific binding

  • Titrate antibody to determine optimal concentration for signal-to-noise ratio

• Insufficient blocking:

  • Inadequate blocking allows non-specific binding

  • Optimize blocking agent (BSA vs. milk) and duration

• Sample degradation:

  • Proteolytic fragments may create unexpected bands

  • Use fresh samples with protease inhibitors

Common causes of false negatives:

• Epitope destruction:

  • Fixation or extraction methods may destroy the epitope

  • Try multiple fixation methods or extraction buffers

• Low expression levels:

  • Target protein may be expressed below detection threshold

  • Use signal amplification methods or more sensitive detection systems

• Incorrect antibody application:

  • Antibody may work for ELISA but not Western blot

  • Validate for each specific application

• Epitope masking:

  • Protein-protein interactions may hide the epitope

  • Try different extraction conditions or epitope retrieval methods

• Batch degradation:

  • Antibody activity may decrease with improper storage

  • Store as recommended (-20°C or -80°C) in small aliquots

For each experiment, include proper controls to distinguish true signals from artifacts. When troubleshooting, change only one variable at a time and document all modifications to protocols .

How can researchers determine the optimal concentration of zgc:153454 antibody for different applications?

Determining optimal antibody concentration requires systematic titration experiments for each application:

• Western Blotting optimization:

  • Prepare serial dilutions (1:500, 1:1000, 1:2000, 1:5000, 1:10000)

  • Use consistent protein amount and identical blotting conditions

  • Evaluate signal-to-noise ratio at each concentration

  • Select concentration that gives specific bands with minimal background

  • Typical working range: 1:1000-1:5000 for most polyclonal antibodies

• Immunohistochemistry/Immunofluorescence optimization:

  • Test dilution series (1:50, 1:100, 1:200, 1:500, 1:1000)

  • Process all samples identically

  • Evaluate specific staining vs. background

  • Include competing peptide control to confirm specificity

  • Typical working range: 1:100-1:500 for tissue sections

• ELISA optimization:

  • Create antibody dilution matrix (1:500 to 1:100,000)

  • Test against different antigen concentrations

  • Generate standard curves at each antibody dilution

  • Select concentration that provides good dynamic range and sensitivity

  • Typical working range: 1:1000-1:10000

• Immunoprecipitation optimization:

  • Test different antibody amounts (1-10μg per reaction)

  • Evaluate pull-down efficiency by Western blot

  • Compare to IgG control for specificity

  • Typical working range: 2-5μg per mg of protein lysate

For each application, plot signal-to-noise ratio against antibody concentration to identify the optimal working range. The goal is to find the lowest concentration that gives reliable detection with minimal background. Document optimization experiments thoroughly for reproducibility .

How should researchers validate zgc:153454 antibody cross-reactivity with human MED13?

Validating potential cross-reactivity between zgc:153454 antibody and human MED13 requires a systematic approach:

• Sequence homology analysis:

  • Perform sequence alignment between zebrafish zgc:153454 and human MED13

  • Focus on the immunogen region used to generate the antibody

  • Calculate percent identity and similarity

  • Identify conserved epitopes that might be recognized

• Western blot validation:

  • Test antibody against human cell lysates

  • Include zebrafish lysate as positive control

  • Look for bands at expected molecular weight of human MED13 (~250 kDa)

  • Confirm specificity using siRNA knockdown of human MED13

• Immunoprecipitation-mass spectrometry:

  • Immunoprecipitate from human lysates using zgc:153454 antibody

  • Identify captured proteins by mass spectrometry

  • Determine if human MED13 is specifically enriched

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Test if this blocks recognition of both zebrafish and human proteins

  • Include human-specific peptide competition as comparison

• Immunofluorescence validation:

  • Test staining pattern in human cells

  • Compare with known localization of human MED13

  • Confirm specificity with siRNA knockdown

Cross-reactivity can be beneficial for comparative studies but must be thoroughly validated. Document the extent of cross-reactivity and any differences in sensitivity between species. Report these findings clearly in publications to guide other researchers .

How can quantitative proteomics approaches be integrated with zgc:153454 antibody research?

Integrating quantitative proteomics with zgc:153454 antibody research enables comprehensive insights into protein function:

• Antibody-based enrichment for targeted proteomics:

  • Use zgc:153454 antibody for immunoprecipitation

  • Process samples for mass spectrometry

  • Identify interaction partners and post-translational modifications

  • Compare interactome across different conditions or developmental stages

• Combined immunoblotting and mass spectrometry:

  • Validate zgc:153454 antibody specificity using immunoprecipitation followed by mass spectrometry

  • Confirm that the antibody captures the intended target protein

  • Identify any cross-reactive proteins

• Parallel reaction monitoring (PRM):

  • Develop targeted mass spectrometry assays for zgc:153454

  • Compare antibody-based quantification with MS-based absolute quantification

  • Use as orthogonal validation method

• SILAC or TMT labeling with immunoenrichment:

  • Implement stable isotope labeling approaches

  • Enrich for zgc:153454 and its complexes using validated antibody

  • Quantify changes in interaction partners or modifications under different conditions

• Absolute quantification using protein standards:

  • Develop quantitative Western blot protocols using purified standards

  • Correlate with absolute quantification by mass spectrometry

  • Generate conversion factors between antibody signal and protein concentration

This integrated approach provides multiple layers of validation and quantitative information about zgc:153454 and its molecular context. When reporting results, clearly describe both antibody-based and MS-based methodologies and highlight concordant findings .

How can researchers determine if discrepancies in zgc:153454 detection are due to technical issues or biological variance?

Distinguishing technical artifacts from biological variance requires systematic investigation:

• Eliminate technical variables first:

  • Repeat experiments using standardized protocols

  • Use the same antibody lot, reagents, and equipment

  • Process all samples simultaneously when possible

  • Include internal reference standards

• Assess biological replicates properly:

  • Use appropriate number of biological replicates (minimum n=3)

  • Calculate coefficient of variation within and between groups

  • Apply appropriate statistical tests based on data distribution

  • Consider power analysis to determine adequate sample size

• Implement control series:

  • Create dilution series of positive control samples

  • Evaluate linearity of detection across concentration range

  • Determine limit of detection and quantification

  • Use to normalize between experiments

• Orthogonal validation:

  • Confirm key findings using alternative detection methods

  • Compare protein levels with mRNA expression

  • Use genetic models (knockdown/knockout) for validation

• Investigate biological sources of variation:

  • Consider developmental timing differences

  • Assess circadian or other temporal effects

  • Evaluate sex-based differences

  • Document age, genetic background, and environmental conditions

• Systematic troubleshooting approach:

  • Change only one variable at a time

  • Document all modifications to protocols

  • Create decision tree for common issues

When reporting results, clearly distinguish between technical variability (error bars on replicates) and biological variability (differences between conditions). Include detailed methods that specify all relevant technical parameters .

What approach should researchers take to study post-translational modifications of zgc:153454?

Studying post-translational modifications (PTMs) of zgc:153454 requires specialized techniques:

• Phosphorylation analysis:

  • Immunoprecipitate zgc:153454 using validated antibody

  • Analyze by Western blot with phospho-specific antibodies

  • Confirm with mass spectrometry using phospho-enrichment

  • Use phosphatase treatment as control

  • Compare phosphorylation status across different conditions

• Ubiquitination detection:

  • Treat samples with proteasome inhibitors to stabilize ubiquitinated proteins

  • Immunoprecipitate with zgc:153454 antibody

  • Blot with anti-ubiquitin antibodies

  • Alternative: express tagged ubiquitin and pull down ubiquitinated proteins

• SUMOylation analysis:

  • Use denaturing conditions for lysis to preserve SUMO modifications

  • Immunoprecipitate zgc:153454

  • Blot with anti-SUMO antibodies

  • Include SUMO protease inhibitors during sample preparation

• Glycosylation detection:

  • Treat samples with glycosidases (PNGase F, O-glycosidase)

  • Observe mobility shifts by Western blot

  • Use lectin binding assays as complementary approach

  • Confirm with mass spectrometry

• Integrated mass spectrometry approach:

  • Enrich for zgc:153454 using immunoprecipitation

  • Process for high-resolution mass spectrometry

  • Use multiple proteases for complete sequence coverage

  • Apply specific enrichment methods for different PTM types

  • Analyze data with appropriate software for PTM identification

• Functional validation of PTMs:

  • Generate site-specific mutants (alanine substitutions)

  • Express in zebrafish embryos and assess functional consequences

  • Use phosphomimetic mutations (S/T to D/E) to study phosphorylation

This comprehensive approach allows for detailed characterization of zgc:153454 PTMs and their functional relevance. Include appropriate controls for each technique and validate key findings using orthogonal methods .