scocb Antibody

What is scocb Antibody and what specific target does it recognize?

The scocb Antibody (Product Code: CSB-PA726386XA01DIL) is a polyclonal antibody raised against recombinant Danio rerio (zebrafish) scocb protein. The target protein is identified by UniProt accession number Q5XJK1. This antibody is developed through immunization with a recombinant form of the zebrafish protein and is purified using antigen affinity methods to enhance specificity . The antibody recognizes epitopes of the native scocb protein in zebrafish samples and has been validated for research applications involving zebrafish models.

What are the optimal storage and handling conditions for scocb Antibody?

Upon receipt, scocb Antibody should be stored at either -20°C or -80°C. To maintain antibody integrity and functionality, it's crucial to avoid repeated freeze-thaw cycles that can lead to protein denaturation and reduced activity . The antibody is provided in a liquid formulation with a storage buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4. This buffer composition helps stabilize the antibody during storage . For routine use, consider preparing small working aliquots to minimize freeze-thaw cycles, as antibody degradation is a common source of experimental variability .

What applications has scocb Antibody been validated for?

The scocb Antibody has been specifically validated for two main applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): Validated for detection and quantification of scocb protein in solution-based assays .

• Western Blotting (WB): Validated for detection of denatured scocb protein in gel electrophoresis applications, providing identification of the antigen based on molecular weight .

For use in other applications such as immunohistochemistry, immunofluorescence, or flow cytometry, additional validation would be necessary as antibodies can perform differently across various applications due to differences in protein conformation and experimental conditions .

How should I validate scocb Antibody before use in my experiments?

Before incorporating scocb Antibody into your research protocol, comprehensive validation is essential to ensure reliable results. Based on established guidelines for antibody validation in physiology studies , follow this methodical approach:

Validation Protocol for scocb Antibody:

• Positive Control Testing:

  • Use zebrafish tissue known to express scocb protein

  • Recombinant scocb protein as a standard reference

  • Compare signal with published expression patterns

• Negative Control Testing:

  • Use tissues from knockout zebrafish (if available)

  • Test in non-expressing tissues

  • Omit primary antibody while maintaining all other conditions

• Specificity Verification:

  • Perform peptide competition assays by pre-incubating antibody with excess antigen

  • Run dilution series (1:500 to 1:10,000) to determine optimal concentration

  • Test with non-immune serum from the same species (rabbit) as control

• Cross-Reactivity Assessment:

  • Test on closely related species if cross-species use is intended

  • Evaluate against related proteins to confirm specificity

Proper validation not only ensures experimental reliability but also aids troubleshooting when unexpected results occur .

What controls are essential when designing experiments with scocb Antibody?

Implementing appropriate controls is critical for reliable interpretation of results when using scocb Antibody. According to guidelines for antibody use in physiology studies , incorporate the following controls:

Required Controls:

• Positive Controls:

  • Known source tissue expressing scocb protein

  • Recombinant scocb protein (if available)

  • Previously validated samples with confirmed expression

• Negative Controls:

  • Tissues from scocb-null zebrafish (if available)

  • Tissues known not to express the target

  • No primary antibody control (to assess secondary antibody specificity)

  • Isotype control (rabbit IgG at equivalent concentration)

• Specificity Controls:

  • Absorption control: Pre-incubate antibody with excess antigen

  • Multiple antibody approach: Use alternative antibodies against the same target

  • Orthogonal validation: Compare with mRNA expression data

• Technical Controls:

  • Loading controls for Western blotting (β-actin, GAPDH)

  • Serial dilution of antibody to determine optimal concentration

  • Serial dilution of antigen to assess sensitivity

  • Secondary antibody-only controls

A comprehensive experimental design should include controls at each stage of the workflow to identify potential sources of error or artifact . Many published studies fail to include adequate controls, with 86% of papers using immunoblot analysis showing no controls, which significantly impacts reproducibility .

What is the recommended protocol for using scocb Antibody in Western blotting?

For optimal Western blotting results with scocb Antibody, follow this detailed protocol based on best practices in antibody research :

Western Blotting Protocol for scocb Antibody:

• Sample Preparation:

  • Extract proteins from zebrafish tissues using RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0)

  • Include protease inhibitors to prevent degradation

  • Quantify protein concentration using BCA or Bradford assay

  • Prepare samples in Laemmli buffer with DTT or β-mercaptoethanol

• Gel Electrophoresis:

  • Load 20-50 μg protein per lane

  • Include molecular weight markers

  • Run SDS-PAGE at 100-120V until appropriate separation

• Transfer:

  • Transfer to PVDF or nitrocellulose membrane (PVDF recommended for higher binding capacity)

  • Use wet transfer at 100V for 1 hour or 30V overnight at 4°C

  • Verify transfer efficiency with Ponceau S staining

• Blocking:

  • Block with 5% non-fat dry milk or BSA in TBS-T for 1 hour at room temperature

  • (BSA recommended if detecting phosphorylated proteins)

• Primary Antibody Incubation:

  • Dilute scocb Antibody 1:1000 in blocking buffer (starting recommendation; optimize as needed)

  • Incubate overnight at 4°C with gentle agitation

• Washing:

  • Wash membrane 3×5 minutes with TBS-T

• Secondary Antibody Incubation:

  • Apply HRP-conjugated anti-rabbit IgG (1:5000 in blocking buffer)

  • Incubate for 1 hour at room temperature

• Detection:

  • Wash membrane 3×5 minutes with TBS-T

  • Apply ECL substrate and detect using appropriate imaging system

  • Capture images before signal saturation

• Data Analysis:

  • Quantify band intensity using densitometry software

  • Normalize to loading control

  • Analyze data from at least three biological replicates

Remember to include appropriate controls with each experiment and show complete blots including molecular weight markers in publications to demonstrate specificity .

How should I quantify and normalize Western blot results obtained with scocb Antibody?

Proper quantification and normalization are essential for reliable interpretation of Western blot data generated with scocb Antibody. Follow these methodological steps:

Quantification Procedure:

• Image Acquisition:

  • Capture images within the linear range of detection

  • Avoid overexposed (saturated) pixels that compress signal differences

  • Use the same exposure settings for all compared samples

  • Acquire 16-bit images when possible for greater dynamic range

• Densitometry Analysis:

  • Use specialized software (ImageJ, Image Studio, etc.) for quantification

  • Draw consistent region of interest (ROI) areas around bands

  • Subtract local background for each band individually

  • Document all quantification parameters for reproducibility

• Normalization Strategies:

  • Normalize to appropriate loading controls (β-actin, GAPDH, or total protein stain)

  • Calculate relative expression as: (Target protein signal / Loading control signal)

  • For phospho-proteins, normalize to total protein levels of the same target

  • Consider housekeeping protein stability under your experimental conditions

What common sources of variability should I control for in scocb Antibody experiments?

Understanding and controlling sources of variability is crucial for obtaining reproducible results with scocb Antibody. Based on antibody research best practices , monitor these key factors:

Major Sources of Experimental Variability:

• Antibody-Related Variability:

  • Lot-to-lot variations (especially critical for polyclonal antibodies like scocb Antibody)

  • Antibody degradation due to improper storage or freeze-thaw cycles

  • Concentration inconsistencies in working solutions

  • Aggregation affecting effective concentration

• Biological Variability:

  • Zebrafish strain differences in target protein expression

  • Developmental stage variations (particularly important in zebrafish research)

  • Sex-dependent expression patterns

  • Individual fish health status and stress levels

  • Environmental conditions during zebrafish rearing

• Technical Variability:

  • Inconsistent sample preparation and protein extraction

  • Variations in protein loading amounts

  • Inconsistent blocking conditions affecting background

  • Temperature fluctuations during incubation

  • Washing stringency differences

  • Detection reagent variability or degradation

Variability Mitigation Strategies:

Research has shown that insufficient method details in 59% of published papers contribute to reproducibility challenges . Detailed documentation of these variables is essential for experimental reproducibility.

What are the implications of using polyclonal scocb Antibody versus potential monoclonal alternatives?

Understanding the fundamental differences between polyclonal antibodies (like scocb Antibody) and monoclonal alternatives is crucial for experimental design and data interpretation:

Comparison of Antibody Types for scocb Research:

Polyclonal scocb Antibody Characteristics:

Advantages:

• Recognizes multiple epitopes on scocb protein, increasing detection sensitivity

• More robust to protein denaturation and fixation conditions

• Better for detecting native proteins with post-translational modifications

• Generally provides stronger signal through cooperative binding

• More tolerant to minor antigen changes or species variations

Limitations:

• Lot-to-lot variability affects experimental reproducibility

• Higher potential for cross-reactivity with related proteins

• Limited supply (serum from one immunized rabbit is finite)

• Less defined specificity makes validation more challenging

Monoclonal Antibody Advantages (if available):

Advantages:

• Consistent specificity between production lots

• Recognizes a single epitope, reducing non-specific binding

• Unlimited supply through hybridoma technology

• Better for distinguishing highly similar proteins

• Easier standardization across experiments and labs

Limitations:

• May miss protein variants with epitope modifications

• Often more sensitive to denaturation or fixation conditions

• Potentially lower sensitivity than polyclonals

• Single epitope recognition may miss some protein forms

Research Implications:

For early characterization of scocb protein function:

• Polyclonal antibodies like the current scocb Antibody provide better initial detection

• For distinguishing between closely related proteins, monoclonals would be preferable

• For quantitative analysis requiring reproducibility across multiple studies, monoclonals offer advantages

• For detecting low abundance proteins, polyclonals typically provide better sensitivity

According to recent developments in antibody technology, recombinant antibodies represent the future direction, as they combine the specificity of monoclonals with improved consistency . One survey found that researchers often need to try 3-4 antibodies before finding one that works reliably for their application .

How can I apply computational approaches to predict scocb Antibody specificity and potential cross-reactivity?

Computational approaches can provide valuable insights into antibody specificity and cross-reactivity before extensive experimental validation. Based on methods described in recent research , consider these approaches:

Computational Prediction Methods:

• Epitope Prediction and Analysis:

  • Use algorithms to predict likely epitopes on scocb protein

  • Compare epitope regions with homologous proteins across species

  • Identify conserved regions that might lead to cross-reactivity

  • Tools like BepiPred, DiscoTope, or ABCpred can predict linear and conformational epitopes

• Sequence Homology Assessment:

  • Perform BLAST analysis of the immunogen sequence used to generate scocb Antibody

  • Identify proteins with high sequence similarity that might cause cross-reactivity

  • Pay special attention to proteins commonly expressed in zebrafish tissues

• Structural Modeling Approaches:

  • Generate structural models of scocb protein using homology modeling

  • Dock antibody binding regions to the target

  • Identify accessible epitopes in native protein conformation

  • Predict effects of mutations or post-translational modifications

• Biophysics-Informed Models:

  • Recent research has developed biophysics-informed models trained on experimentally selected antibodies

  • These models can associate potential ligands with distinct binding modes

  • Such approaches can predict antibody specificity beyond experimentally observed variants

Research has demonstrated that computational approaches can successfully predict antibody solubility and specificity . For example, one study used CamSol to predict monoclonal antibody solubility with high accuracy, allowing researchers to select more promising antibody candidates early in development .

How can I determine if scocb Antibody recognizes post-translational modifications of the target protein?

Post-translational modifications (PTMs) can significantly impact antibody recognition and experimental results. To determine whether scocb Antibody recognizes modified forms of the target protein, implement this systematic approach:

Methodological Strategy:

• Literature and Database Analysis:

  • Research known PTMs of scocb protein (phosphorylation, glycosylation, etc.)

  • Check UniProt entry Q5XJK1 for annotated modification sites

  • Evaluate if the antibody's immunogen sequence contains potential modification sites

• Experimental Approaches:

  • Phosphorylation Analysis:

    • Treat samples with phosphatase enzymes before Western blotting

    • Compare migration patterns before and after treatment

    • Use phospho-specific detection methods (e.g., Phos-tag gels) for comparison

  • Glycosylation Analysis:

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

    • Observe changes in molecular weight or signal intensity

    • Compare with lectin blotting to confirm glycosylation status

  • Other Modifications:

    • Use specific inhibitors of modification processes in cell/tissue preparation

    • Compare samples from different conditions known to affect modification status

    • Immunoprecipitate with scocb Antibody and analyze by mass spectrometry

• Validation with Modified Protein Standards:

  • Use recombinant scocb protein with defined modification status as controls

  • Compare antibody recognition of modified vs. unmodified forms

  • Create a panel of samples with varying modification levels for calibration

Analytical Framework:

Research on antibody performance has shown that recognition of post-translational modifications can significantly affect experimental results and interpretation, particularly when antibodies have been raised against unmodified recombinant proteins . Understanding these interactions is crucial for accurate data interpretation.

What are the best practices for reporting scocb Antibody usage in scientific publications?

Proper reporting of antibody usage is essential for experimental reproducibility. Based on guidelines for antibody citation , adhere to these best practices when reporting scocb Antibody usage in publications:

Essential Reporting Elements:

• Complete Antibody Identification:

  • Full product name: anti-scocb polyclonal antibody

  • Manufacturer: Cusabio

  • Catalog number: CSB-PA726386XA01DIL

  • Lot number (critical for polyclonal antibodies)

  • RRID (Research Resource Identifier) if available

• Validation Information:

  • How you validated the antibody for your specific application

  • Controls used to verify specificity

  • References to previous validation studies if applicable

• Detailed Methodology:

  • Sample preparation procedures in detail

  • Dilution factor used (e.g., 1:1000)

  • Incubation conditions (time, temperature, buffer composition)

  • Detection method and settings

  • Image acquisition parameters

• Results Presentation:

  • Show representative blots/images with molecular weight markers

  • Include both positive and negative controls

  • Present quantification from multiple biological replicates (n≥3)

  • Clearly indicate any image processing performed

Example Materials and Methods Section:

"Western blotting was performed using rabbit anti-scocb polyclonal antibody (Cusabio, Cat# CSB-PA726386XA01DIL, Lot# 123456) at 1:1000 dilution in 5% BSA in TBST. The antibody specificity was validated by peptide competition assay and testing in scocb-/- samples. Membranes were incubated overnight at 4°C followed by HRP-conjugated goat anti-rabbit secondary antibody (Vendor, Cat#) at 1:5000 for 1 hour at room temperature. Signals were detected using enhanced chemiluminescence and imaged using [equipment details]. Band intensities were quantified using ImageJ software (version X) and normalized to β-actin levels. Each experiment was performed with n=3 biological replicates."

Comprehensive Publication Checklist:

Studies have shown that less than half of antibodies used in publications can be identified, making reproduction difficult . Comprehensive reporting addresses this concern and enhances scientific rigor.

How do I address weak or absent signal when using scocb Antibody in Western blotting?

When facing weak or absent signals with scocb Antibody in Western blotting, employ this systematic troubleshooting approach:

Root Cause Analysis and Solutions:

• Antibody-Related Issues:

  • Problem: Antibody degradation or denaturation

  • Solution: Use fresh aliquot, verify storage conditions

  • Verification: Test with positive control known to work previously

  • Problem: Insufficient antibody concentration

  • Solution: Titrate antibody (try 1:500, 1:200, or even 1:100)

  • Verification: Compare signal intensities across dilution series

• Sample-Related Issues:

  • Problem: Low target protein abundance

  • Solution: Increase protein loading (50-100 μg)

  • Verification: Verify protein transfer with Ponceau S staining

  • Problem: Protein degradation

  • Solution: Add fresh protease inhibitors, keep samples cold

  • Verification: Check for degradation bands or smears

  • Problem: Inefficient protein extraction

  • Solution: Try different lysis buffers (RIPA, NP-40, etc.)

  • Verification: Validate extraction with known abundant proteins

• Protocol-Related Issues:

  • Problem: Inefficient transfer

  • Solution: Optimize transfer conditions (time, voltage)

  • Verification: Use pre-stained markers to confirm transfer

  • Problem: Excessive blocking

  • Solution: Reduce blocking time or concentration

  • Verification: Try different blocking agents (milk, BSA, commercial blockers)

  • Problem: Incompatible detection method

  • Solution: Try more sensitive detection (ECL-Plus, femto reagents)

  • Verification: Ensure substrate is fresh and properly prepared

Systematic Troubleshooting Approach:

Research in antibody validation shows that optimization requirements are substantial, with researchers often needing to try multiple dilutions and conditions before achieving reliable results . This is especially true for antibodies against novel or less-studied targets like scocb.

How can I develop a customized ELISA for scocb protein quantification using this antibody?

Developing a customized ELISA for scocb protein quantification requires careful optimization. Follow this detailed methodological approach:

ELISA Development Protocol:

• Plate Coating Optimization:

  • Direct Coating Method:

    • Coat wells with recombinant scocb protein at 1-10 μg/ml in carbonate buffer (pH 9.6)

    • Establish standard curve with 2-fold serial dilutions

    • Incubate overnight at 4°C

  • Capture Antibody Method (if second antibody available):

    • Coat with anti-scocb antibody (1-10 μg/ml) in carbonate buffer

    • Block and add samples containing target protein

    • Detect with biotinylated detection antibody

• Blocking Optimization:

  • Test different blocking agents:

    • 1-5% BSA in PBS

    • 1-5% non-fat dry milk in PBS

    • Commercial blocking buffers

  • Optimal blocking: 1 hour at room temperature

• Antibody Dilution Optimization:

  • Create dilution matrix for scocb Antibody:

    • Range: 1:100 to 1:10,000

    • Test against different antigen concentrations

    • Select dilution with best signal-to-noise ratio

• Detection System Optimization:

  • Compare detection methods:

    • HRP-conjugated secondary + TMB substrate

    • Biotin-streptavidin amplification

    • Fluorescent detection

  • Optimize development time: typically 5-30 minutes

• Validation Steps:

  • Determine detection limits using known standards

  • Evaluate precision with replicate measurements

  • Assess specificity through competition assays

  • Confirm linearity within working range

Method Development Table:

Validation and Quality Control:

After optimization, validate your assay by measuring:

• Intra-assay CV (coefficient of variation): Target <10%

• Inter-assay CV: Target <15%

• Recovery of spiked samples: Target 80-120%

• Parallelism between standard and sample dilution curves