tam13 Antibody

What is tam13 Antibody and what are its basic characteristics?

tam13 Antibody (product code CSB-PA516837XA01SXV) is a polyclonal antibody raised in rabbits against recombinant Schizosaccharomyces pombe (strain 972/ATCC 24843) tam13 protein. It is provided in liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative. The antibody targets the protein corresponding to UniProt accession number G2TRQ0 and has been validated for ELISA and Western blot applications .

When working with this antibody, it's essential to understand that it's designed specifically for research use in fission yeast systems, not for diagnostic or therapeutic applications. The antibody recognizes epitopes on the tam13 protein, which serves as an important experimental tool for researchers studying S. pombe cellular processes.

What are the recommended storage conditions for tam13 Antibody?

For optimal longevity and performance, tam13 Antibody should be stored at either -20°C or -80°C upon receipt. It's critical to avoid repeated freeze-thaw cycles as these can significantly degrade antibody activity and reduce specificity. Since the antibody is supplied in a glycerol-containing buffer (50% glycerol), it remains liquid at freezer temperatures, facilitating aliquoting .

When handling the antibody, best practices include:

• Aliquoting the stock into single-use volumes to minimize freeze-thaw cycles

• Always keeping the antibody on ice when in use

• Briefly centrifuging tubes after thawing to collect all liquid at the bottom

• Recording lot numbers and purchase dates for experimental reproducibility

• Following manufacturer guidelines for recommended shelf-life

What applications has tam13 Antibody been validated for?

tam13 Antibody has been specifically tested and validated for enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) applications. These techniques allow researchers to detect and quantify tam13 protein in research samples .

What is the optimal protocol for using tam13 Antibody in Western blotting?

For optimal Western blot results with tam13 Antibody, the following methodological approach is recommended:

• Sample Preparation:

  • Harvest S. pombe cells at mid-log phase

  • Lyse cells in buffer containing protease inhibitors

  • Quantify protein concentration using Bradford or BCA assay

• Gel Electrophoresis:

  • Load 20-50 µg total protein per lane on 10-12% SDS-PAGE

  • Include positive and negative controls

• Transfer and Blocking:

  • Transfer to PVDF membrane (0.45 µm pore size)

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Antibody Incubation:

  • Dilute tam13 Antibody 1:500 to 1:2000 in blocking buffer

  • Incubate membrane overnight at 4°C with gentle agitation

  • Wash 3× with TBST (10 minutes each)

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour

  • Wash 3× with TBST (10 minutes each)

• Detection:

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Expected molecular weight for tam13 protein is approximately 45-50 kDa

This protocol should be optimized for specific experimental conditions, and results should be validated using appropriate controls to ensure specificity .

How can researchers troubleshoot weak or absent signals when using tam13 Antibody?

When encountering weak or absent signals with tam13 Antibody, a systematic troubleshooting approach is essential:

For persistent issues, consider experimenting with alternative detection methods or confirming target protein expression under your specific experimental conditions. When working with yeast samples, cell wall disruption efficiency can significantly impact protein extraction yield, so optimizing lysis conditions may improve results .

How can specificity of tam13 Antibody be validated in research applications?

Validating the specificity of tam13 Antibody requires a multi-faceted approach:

• Genetic Validation:

  • Compare signal between wild-type and tam13 knockout/knockdown S. pombe strains

  • Use overexpression systems to confirm signal intensification

  • Generate epitope-tagged versions for parallel detection with tag-specific antibodies

• Molecular Validation:

  • Confirm single band of expected molecular weight in Western blot

  • Perform mass spectrometry analysis of immunoprecipitated material

  • Use peptide competition assays to confirm epitope specificity

• Cross-Reactivity Assessment:

  • Test against related yeast species lacking tam13 homologs

  • Evaluate reactivity with recombinant tam13 fragments to map epitopes

  • Document any cross-reacting proteins

• Technical Controls:

  • Include isotype control antibodies raised in the same species

  • Use secondary-only controls to assess non-specific binding

  • Test pre-immune serum when available

Similar validation approaches have been widely used for antibodies targeting proteins in the TAM receptor family, demonstrating that comprehensive validation is essential for confident interpretation of research findings .

How can tam13 Antibody be optimized for immunoprecipitation experiments?

While primarily validated for ELISA and Western blot, tam13 Antibody can be adapted for immunoprecipitation with methodological adjustments:

• Lysis Buffer Optimization:

  • Use mild, non-denaturing buffer to preserve protein conformation

  • Include protease inhibitors, phosphatase inhibitors, and EDTA

  • Test different detergent concentrations (0.5-1% NP-40 or Triton X-100)

• Pre-clearing Strategy:

  • Pre-clear lysate with Protein A/G beads (1 hour at 4°C)

  • Remove non-specific binding proteins by centrifugation

  • Retain a sample of pre-cleared lysate as input control

• Antibody Binding:

  • Use 2-5 µg antibody per 500 µg total protein

  • Incubate overnight at 4°C with gentle rotation

  • Add pre-washed Protein A/G beads and incubate 2-4 hours

• Washing and Elution:

  • Use increasingly stringent washes to reduce background

  • Elute bound proteins with low pH buffer or SDS sample buffer

  • Analyze by Western blot or mass spectrometry

• Controls:

  • Include negative control (normal rabbit IgG)

  • Use tam13 knockout samples as specificity controls

  • Verify immunoprecipitation efficiency by immunoblotting

Similar immunoprecipitation approaches have been successfully employed with antibodies targeting other TAM family proteins, suggesting this methodology can be adapted for tam13 .

What strategies can be used to adapt tam13 Antibody for fluorescence microscopy?

Adapting tam13 Antibody for immunofluorescence microscopy requires special considerations for yeast cells:

• Cell Wall Digestion:

  • Treat cells with enzymatic cocktail (zymolyase/lyticase) to create spheroplasts

  • Optimize digestion time to balance cell integrity and antibody accessibility

  • Monitor spheroplast formation microscopically

• Fixation Optimization:

  • Test multiple fixation methods:

    • 4% paraformaldehyde (10-20 minutes)

    • Cold methanol (-20°C, 6 minutes)

    • Combined formaldehyde-methanol fixation

  • Balance epitope preservation with membrane permeabilization

• Antibody Incubation:

  • Block with 3-5% BSA in PBS (30-60 minutes)

  • Test tam13 Antibody at 1:100 to 1:500 dilutions

  • Incubate overnight at 4°C in humidity chamber

  • Use fluorophore-conjugated anti-rabbit secondary antibody (1:500-1:1000)

• Imaging Controls:

  • Include tam13 knockout strains as negative controls

  • Use DAPI for nuclear counterstaining

  • Acquire Z-stack images to capture the entire cell volume

  • Compare signal distribution with known tam13 localization data

This approach incorporates methodological principles successfully applied to similar antibodies in yeast systems and can be refined based on specific experimental needs .

Can tam13 Antibody be used in chromatin immunoprecipitation (ChIP) studies?

Using tam13 Antibody for ChIP requires specific optimization strategies:

• Cross-linking Protocol:

  • Test formaldehyde concentrations (0.75-1.5%)

  • Optimize cross-linking time (5-20 minutes)

  • Quench with glycine (125 mM final concentration)

• Chromatin Preparation:

  • For S. pombe, use specialized protocols for spheroplasting

  • Optimize sonication conditions to achieve 200-500 bp fragments

  • Verify fragmentation by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Use 2-10 μg tam13 Antibody per reaction

  • Include IgG control and input samples

  • Incubate overnight at 4°C with rotation

• Washing and Elution:

  • Use increasingly stringent wash buffers

  • Elute protein-DNA complexes with SDS buffer

  • Reverse cross-links (65°C, 6-12 hours)

  • Purify DNA for downstream analysis

• Validation Analysis:

  • Perform qPCR for suspected binding regions

  • Compare enrichment to input and IgG controls

  • Verify specificity using tam13 knockout controls

Similar approaches have been successfully employed with antibodies targeting TAM family proteins in other contexts, suggesting this methodology could be adapted for tam13 in appropriate experimental systems .

What quantitative approaches are recommended for analyzing Western blot data from tam13 Antibody experiments?

Rigorous quantification of Western blot data requires methodological precision:

• Experimental Design for Quantification:

  • Include calibration standards (recombinant tam13 protein)

  • Use technical and biological replicates (minimum n=3)

  • Include proper loading controls (housekeeping proteins)

• Image Acquisition Parameters:

  • Capture images within linear dynamic range

  • Avoid pixel saturation (check histogram)

  • Maintain consistent exposure settings across samples

  • Consider using fluorescent secondary antibodies for wider linear range

• Densitometry Analysis:

  • Use specialized software (ImageJ, Image Lab)

  • Define analysis regions consistently

  • Subtract local background for each lane

  • Normalize to loading controls or total protein

• Statistical Analysis:

  • Apply appropriate statistical tests based on experimental design

  • Report both means and measures of variability (SD or SEM)

  • Use non-parametric tests for non-normally distributed data

  • Apply correction for multiple comparisons when appropriate

These quantitative approaches ensure reliable data interpretation and have been successfully applied in studies using antibodies against similar target proteins .

How should researchers interpret variations in tam13 protein expression across different experimental conditions?

Interpreting variations in tam13 protein expression requires methodological considerations:

• Normalization Strategies:

  • Normalize to multiple housekeeping proteins, not just one

  • Consider total protein normalization using stain-free technology

  • Validate stability of reference proteins under your experimental conditions

• Biological Context:

  • Compare expression changes with mRNA levels when possible

  • Consider post-translational modifications affecting antibody recognition

  • Evaluate protein half-life and turnover rates in your system

• Statistical Analysis:

  • Establish thresholds for biological significance beyond statistical significance

  • Consider power analysis to determine appropriate sample sizes

  • Use fold-change rather than absolute values for comparisons

• Validation Approaches:

  • Confirm key findings with alternative detection methods

  • Perform time-course experiments to capture dynamic changes

  • Correlate protein levels with functional outcomes

Similar analytical approaches have been employed in studies examining expression of TAM family proteins across different experimental conditions, providing a methodological framework that can be applied to tam13 .

How can tam13 Antibody be integrated into multi-omics research approaches?

Integrating tam13 Antibody into multi-omics research requires coordinated experimental design:

• Sample Preparation Coordination:

  • Develop protocols allowing parallel extraction of protein, RNA, and DNA

  • Use consistent growth conditions across all analyses

  • Consider sequential extraction methods for limited samples

• Integration with Transcriptomics:

  • Correlate tam13 protein levels with mRNA expression

  • Design time-course experiments to capture regulatory dynamics

  • Compare wild-type with genetically modified strains

• Integration with Proteomics:

  • Use tam13 Antibody for immunoprecipitation coupled with mass spectrometry

  • Identify interaction partners under different conditions

  • Compare post-translational modifications with protein function

• Integration with Genomics:

  • Use ChIP-seq to identify tam13 binding sites if applicable

  • Correlate binding with gene expression changes

  • Analyze effects of genetic variants on tam13 function

• Data Integration Framework:

  • Apply computational approaches to integrate multi-omics datasets

  • Use network analysis to identify functional relationships

  • Develop predictive models of tam13 function in cellular processes

Similar integrative approaches have been successfully applied with antibodies targeting other proteins, particularly in the TAM receptor family, demonstrating the value of multi-omics integration .

What emerging technologies might enhance the research applications of tam13 Antibody?

Emerging technologies offer new opportunities for tam13 Antibody applications:

• Advanced Imaging Technologies:

  • Super-resolution microscopy for precise localization

  • Live-cell imaging with antibody fragments

  • Expansion microscopy for enhanced spatial resolution

  • Correlative light and electron microscopy

• Single-Cell Analysis:

  • Mass cytometry (CyTOF) for high-parameter protein analysis

  • Microfluidic antibody capture for single-cell proteomics

  • Spatial transcriptomics combined with protein detection

• Proximity Labeling Technologies:

  • Antibody-directed enzyme-mediated proximity labeling

  • Identification of proximal proteins in native cellular contexts

  • Mapping protein microenvironments

• Synthetic Biology Applications:

  • Antibody-directed protein degradation systems

  • Optogenetic control of antibody-based detection

  • Creation of biosensors using antibody fragments

• AI-Enhanced Antibody Applications:

  • Machine learning for epitope prediction and optimization

  • Automated image analysis for complex phenotyping

  • Structure prediction for antibody-antigen interactions

Recent advances in AI-based antibody design demonstrate how computational approaches can enhance antibody development and application, a principle that could be extended to tam13 Antibody research .