ST13 PAT5C6AT Antibody

What is ST13 protein and why is it important in research?

ST13 (Suppression of Tumorigenicity 13) is an adaptor protein or co-chaperone that mediates the association between HSP70 and HSP90 and appears in early receptor complexes. Through common binding to both Hsp70 and Hsp90, ST13 functions as an adaptor that can integrate Hsp70 and Hsp90 interactions. ST13 plays a significant role in the assembly process of the glucocorticoid receptor, requiring the assistance of multiple molecular chaperones to achieve proper functioning. The protein has gained research importance because its expression is downregulated in colorectal carcinoma tissue compared to adjacent normal tissue, suggesting its role as a candidate tumor suppressor gene. The expression levels of the ST13 gene are significantly decreased in primary tumors compared with adjacent mucosa, making it an important research target in cancer biology .

What are the key characteristics of the ST13 PAT5C6AT antibody?

The ST13 PAT5C6AT antibody is a mouse anti-human monoclonal antibody derived from the hybridization of mouse F0 myeloma cells with spleen cells from BALB/c mice immunized with recombinant human ST13 protein (amino acids 1-369) purified from E. coli. It belongs to the IgG2a isotype with kappa light chain. The antibody has been purified from mouse ascitic fluids using protein-A affinity chromatography, ensuring high purity and specificity. It primarily reacts with human samples, though some versions may also show reactivity with mouse and rat samples .

What are the common applications for ST13 PAT5C6AT antibody?

The ST13 PAT5C6AT antibody has been validated for multiple research applications including:

• Western Blot (WB): Typically used at dilutions of 1:1000-1:6000

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunofluorescence (IF)

• Immunocytochemistry (ICC)

• Immunohistochemistry (IHC): Recommended dilutions of 1:200-1:1000

• Flow Cytometry

• Immunoprecipitation (IP): Using 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

The antibody has been tested in various cell lines and tissue samples, including HEK-293 cells, human colon cancer tissue, and mouse testis tissue, demonstrating its versatility for different experimental contexts .

How can I optimize Western blot protocols for ST13 detection?

For optimal Western blot results with ST13 PAT5C6AT antibody, consider the following methodological approach:

• Sample preparation: Use 30 μg of protein under reducing conditions. The antibody has been validated with various sample types including human cell lines (HeLa, A431, 293T, SK-OV-3), rat tissues (testis, C6 cells), and mouse samples (testis, NIH/3T3 cells).

• Gel electrophoresis: Use a 5-20% SDS-PAGE gradient gel for optimal separation. Run at 70V for the stacking gel and 90V for the resolving gel, with a total run time of 2-3 hours.

• Protein transfer: Transfer proteins to a nitrocellulose membrane at 150 mA for 50-90 minutes.

• Blocking: Block the membrane with 5% non-fat milk in TBS for 1.5 hours at room temperature.

• Primary antibody incubation: Use ST13 antibody at 0.25 μg/mL (approximately 1:1000-1:6000 dilution) and incubate overnight at 4°C.

• Washing: Wash with TBS-0.1% Tween three times, 5 minutes each.

• Secondary antibody: Incubate with goat anti-rabbit or anti-mouse IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hours at room temperature.

• Detection: Develop signal using an Enhanced Chemiluminescent detection kit.

The expected band size for ST13 is approximately 41 kDa, though it may appear between 45-54 kDa on some gels due to post-translational modifications .

How does ST13 expression vary across cancer types, and what are the implications for using this antibody in cancer research?

ST13 expression shows significant tissue-specific patterns in cancer, particularly in colorectal cancer, where its downregulation has been well-documented. The ST13 protein is mostly expressed in colorectal epithelia and adenocarcinoma cells, with expression levels significantly decreased in primary tumors compared to adjacent normal mucosa. This downregulation pattern suggests ST13 functions as a tumor suppressor.

When designing cancer research studies utilizing the ST13 PAT5C6AT antibody, researchers should consider:

• Tissue-specific expression: Include appropriate positive controls from tissues known to express ST13, such as normal colorectal epithelia.

• Comparative analysis: Design experiments that compare expression levels between tumor and adjacent normal tissue to identify differential expression patterns.

• Functional studies: Consider complementing expression studies with functional assays to investigate how ST13 downregulation affects HSP70/HSP90 chaperone function and the downstream cellular processes they regulate.

• Correlation with clinical parameters: Analyze ST13 expression in relation to tumor grade, stage, and patient outcomes to determine its prognostic value.

This antibody has been validated for immunohistochemistry in human colon cancer tissue, making it suitable for translational cancer research applications .

What controls should be included in immunofluorescence experiments using the ST13 PAT5C6AT antibody?

When performing immunofluorescence with ST13 PAT5C6AT antibody, appropriate controls are essential for result validation. Three main control types should be included:

• Positive controls:

  • Include a sample known to express ST13 (such as HeLa cells or colorectal epithelia)

  • Use an antibody against a housekeeping protein (like β-actin or GAPDH) in a parallel experiment to confirm sample quality

• Endogenous controls to assess cell/tissue health:

  • Include markers to check for cell stress (phospho-histone H2AX for DNA damage)

  • Examine mitochondrial morphology for stress indicators

  • For tissue samples, check for signs of cell death using antibodies against cleaved caspase or PARP

  • Look for morphological changes like condensed nuclei or cell blebbing

  • Holes in tissue architecture may indicate improper storage or embedding of samples

• Negative controls:

  • Secondary antibody-only control to assess non-specific background staining

  • PBS-only treatment to evaluate autofluorescence

  • Isotype control (using the same IgG subclass as the ST13 antibody but with no specific target)

These controls will help set appropriate maximum exposure and laser power settings to avoid non-specific signal and ensure confidence that the antibody is binding specifically to ST133 .

How can I optimize immunohistochemistry (IHC) protocols for ST13 detection in tissue samples?

For optimal IHC detection of ST13 in tissue samples using the PAT5C6AT antibody, follow these methodological steps:

• Sample preparation:

  • Use properly fixed tissue sections (typically formalin-fixed, paraffin-embedded)

  • For frozen sections, ensure proper tissue embedding and sectioning to maintain morphology

• Antigen retrieval:

  • Primary recommendation: Use TE buffer at pH 9.0

  • Alternative method: Citrate buffer at pH 6.0

  • Heat-induced epitope retrieval methods are typically more effective than proteolytic methods for ST13

• Blocking:

  • Block with 10% normal goat serum to reduce non-specific binding

  • Include additional blocking steps if tissue contains high levels of endogenous peroxidase

• Antibody dilution and incubation:

  • Use ST13 PAT5C6AT antibody at 1:200-1:1000 dilution

  • Incubate overnight at 4°C for optimal results

  • For sensitive detection, consider using amplification systems such as biotin-streptavidin

• Detection system:

  • Use appropriate secondary antibodies compatible with the host species (anti-mouse)

  • For colorimetric detection, DAB (3,3'-diaminobenzidine) provides good contrast

  • For fluorescent detection, consider using Cy3-conjugated secondary antibodies

• Counterstaining and mounting:

  • Hematoxylin counterstain for brightfield microscopy

  • DAPI for fluorescence applications

  • Use appropriate mounting media based on detection method (aqueous for fluorescence)

• Parallel controls:

  • Run known positive tissue (colon cancer tissue has been validated)

  • Include negative controls (no primary antibody, isotype control)

Remember that optimal dilutions may vary based on tissue type and fixation method, so preliminary titration experiments are recommended to determine the optimal antibody concentration for specific experimental conditions .

What are common challenges in Western blot detection of ST13 and how can they be resolved?

When working with ST13 PAT5C6AT antibody in Western blot applications, researchers may encounter several challenges:

• Multiple bands or unexpected molecular weight:

  • Expected molecular weight of ST13 is 41 kDa, but it may appear between 45-54 kDa

  • Solution: Use positive control lysates (HEK-293, HeLa cells) with known ST13 expression

  • Consider running gradient gels (5-20% SDS-PAGE) for better resolution

  • Post-translational modifications may cause shifts in apparent molecular weight

• Weak or no signal:

  • Increase antibody concentration (try 1:1000 instead of 1:6000)

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

  • Ensure protein transfer efficiency with Ponceau S staining

  • Verify sample preparation methods preserve protein integrity

  • Check if sample buffer contains appropriate reducing agents

• High background:

  • Increase blocking time or concentration (try 5% BSA instead of milk)

  • Add 0.1-0.3% Tween-20 to washing buffer

  • Decrease secondary antibody concentration

  • Ensure membrane is completely covered during incubation steps

  • Use freshly prepared buffers

• Non-specific bands:

  • Increase wash duration and frequency after primary and secondary antibody incubations

  • Try alternative blocking reagents (BSA, casein, commercial blocking buffers)

  • Validate with knockout or knockdown samples if available

  • Consider pre-adsorption of antibody with recombinant protein

For optimal results, ST13 antibody has been successfully used with multiple human cell lines (HeLa, A431, 293T, SK-OV-3) and mouse/rat samples (testis tissues, NIH/3T3, C6 cells) at concentrations of 0.25 μg/mL, with overnight incubation at 4°C .

How can I differentiate between true ST13 signal and artifacts in immunofluorescence experiments?

Distinguishing genuine ST13 signal from artifacts in immunofluorescence experiments requires careful experimental design and systematic controls:

• Subcellular localization assessment:

  • ST13 as a co-chaperone typically shows cytoplasmic localization

  • Unusual localization patterns (such as exclusively nuclear) may indicate non-specific binding

  • Co-stain with markers of cellular compartments to confirm expected localization

• Signal intensity evaluation:

  • Genuine ST13 staining should show dose-dependent response with antibody concentration

  • Compare staining patterns between different cell types with known varying ST13 expression levels

  • Excessive brightness throughout all cells regardless of expression level suggests non-specific binding

• Pattern consistency:

  • Compare with published literature on ST13 localization

  • Artifact staining often appears as:

    • Even staining across all cell types regardless of expected expression differences

    • Staining restricted to cell boundaries or nuclei only

    • Punctate pattern that doesn't correlate with expected distribution

• Control experiments:

  • Secondary antibody-only controls help identify background staining

  • Competitive blocking with recombinant ST13 protein should reduce specific signal

  • Knockdown validation using siRNA against ST13 should diminish signal proportionally

  • Peptide competition assays can confirm antibody specificity

• Technical considerations:

  • Use appropriate fixation methods (4% paraformaldehyde works well for ST13)

  • Permeabilize cells adequately (0.1% Triton X-100 is typically effective)

  • Block with 10% normal goat serum to reduce non-specific binding

  • Use optimized antibody concentration (5 μg/mL has been validated)

  • Counter-stain nuclei with DAPI to aid in cellular orientation

For validated IF protocol, successful staining has been demonstrated in HeLa cells using enzyme antigen retrieval, followed by blocking with 10% goat serum, incubation with 5 μg/mL ST13 PAT5C6AT antibody overnight at 4°C, and detection with Cy3-conjugated secondary antibody3 .

How can the ST13 PAT5C6AT antibody be used to study protein-protein interactions involving the HSP70/HSP90 chaperone system?

The ST13 PAT5C6AT antibody can be effectively utilized to investigate protein-protein interactions within the HSP70/HSP90 chaperone complex through several methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use 0.5-4.0 μg of ST13 antibody per 1.0-3.0 mg of protein lysate

  • Perform pull-down experiments to identify binding partners of ST13

  • Follow with Western blot analysis to detect HSP70, HSP90, or other suspected interacting proteins

  • This approach can reveal how ST13 functions as an adaptor between HSP70 and HSP90

• Proximity Ligation Assay (PLA):

  • Combine ST13 PAT5C6AT antibody with antibodies against potential interacting partners

  • This technique allows visualization of protein-protein interactions in situ with high sensitivity

  • Quantify interaction signals under different cellular conditions or treatments

• Immunofluorescence co-localization:

  • Use dual immunofluorescence with ST13 PAT5C6AT antibody and antibodies against HSP70 or HSP90

  • Analyze co-localization coefficients using appropriate software

  • This approach works particularly well in HeLa cells where ST13 antibody has been validated

• FRET analysis:

  • Combine immunofluorescence using ST13 PAT5C6AT antibody with fluorescently tagged HSP70/HSP90

  • Measure energy transfer to assess proximity of proteins in living cells

  • This technique provides spatial information about chaperone complex assembly

• Chromatin immunoprecipitation (ChIP):

  • For studying ST13's role in glucocorticoid receptor complex formation and DNA binding

  • Use ST13 antibody to pull down protein-DNA complexes

  • Analyze whether ST13 is present at specific genomic loci during transcriptional regulation

These approaches can be particularly valuable for understanding how ST13 downregulation in cancer affects chaperone complex formation and function, potentially providing insights into novel therapeutic approaches targeting the HSP70/HSP90 chaperone system .

What quantitative approaches can be used to measure ST13 expression levels in normal versus cancer tissues?

To accurately measure differential ST13 expression between normal and cancer tissues, researchers can employ several quantitative approaches using the ST13 PAT5C6AT antibody:

• Western blot densitometry:

  • Perform Western blot using standardized amounts of protein from matched normal/tumor samples

  • Use 1:1000-1:6000 dilution of ST13 PAT5C6AT antibody

  • Quantify band intensity using densitometry software

  • Normalize to loading controls such as β-actin or GAPDH

  • This method provides semi-quantitative comparison of protein levels

• Quantitative immunohistochemistry (IHC):

  • Perform IHC with ST13 PAT5C6AT antibody at 1:200-1:1000 dilution on tissue microarrays

  • Use digital pathology software to quantify:

    • Staining intensity (0-3+ scale)

    • Percentage of positive cells

    • H-score (intensity × percentage)

    • Allred score or other standardized scoring systems

  • Compare scores between normal epithelia and matched tumor samples

  • This approach preserves tissue architecture and allows spatial analysis

• Flow cytometry:

  • Prepare single-cell suspensions from fresh tissue or cell lines

  • Fix with 4% paraformaldehyde and permeabilize cells

  • Stain with ST13 PAT5C6AT antibody (1 μg per 1×10^6 cells)

  • Use fluorophore-conjugated secondary antibody

  • Measure median fluorescence intensity (MFI) as a quantitative measure of expression

  • This method allows analysis of ST13 expression in specific cell populations

• Multiplexed protein assays:

  • Develop custom protein arrays with ST13 capture antibody

  • Quantify using standard curves

  • This approach allows simultaneous measurement of multiple proteins

  • Useful for correlating ST13 levels with other chaperone proteins

• Mass spectrometry-based proteomics:

  • Use ST13 antibody for immunoprecipitation followed by mass spectrometry

  • Allows absolute quantification of ST13 and interacting partners

  • Can detect post-translational modifications

When analyzing colorectal cancer samples, studies have consistently shown decreased ST13 expression in tumor tissue compared to adjacent normal mucosa. Quantitative analysis can help establish whether this downregulation correlates with clinical parameters such as tumor stage, grade, or patient outcomes .

What are the key considerations for experimental reproducibility when using ST13 PAT5C6AT antibody?

Ensuring reproducible results when working with ST13 PAT5C6AT antibody requires attention to several critical factors:

• Antibody validation and characterization:

  • Verify antibody specificity using positive and negative controls

  • Document the exact clone (PAT5C6AT), host species (mouse), and isotype (IgG2a)

  • Note the specific epitope recognized (human ST13 protein, amino acids 1-369)

  • Maintain detailed records of antibody lot numbers as performance may vary between batches

• Standardized protocols:

  • Develop detailed standard operating procedures (SOPs) for each application

  • Document critical parameters like antibody dilutions (1:1000-1:6000 for WB, 1:200-1:1000 for IHC)

  • Maintain consistent incubation times and temperatures (overnight at 4°C for primary antibody)

  • Use the same detection systems across experiments

  • For WB, standardize protein loading amounts (30 μg recommended)

• Sample handling and preparation:

  • Use consistent sample collection and processing methods

  • Standardize lysis buffers and protein extraction protocols

  • For tissue samples, maintain consistent fixation times and conditions

  • Document storage conditions and freeze-thaw cycles of antibody and samples

• Quantification methods:

  • Use consistent image acquisition settings

  • Apply standardized quantification methods (densitometry for WB, scoring systems for IHC)

  • Include calibration standards where possible

  • Document software and analysis parameters used

• Experimental controls:

  • Include positive controls (HEK-293 cells, HeLa cells, colon tissue)

  • Use negative controls (secondary antibody only, isotype controls)

  • Include loading controls for normalization in quantitative experiments

  • Consider using siRNA knockdown or CRISPR knockout samples as gold-standard controls

• Data reporting:

  • Report complete experimental conditions in publications

  • Include representative images of controls

  • Share raw data when possible

  • Document any deviations from standard protocols

By addressing these factors systematically, researchers can significantly improve the reproducibility of experiments using the ST13 PAT5C6AT antibody across different laboratory settings and experimental conditions .

What are emerging research areas where ST13 antibodies may provide valuable insights?

The ST13 PAT5C6AT antibody has potential applications in several emerging research areas that extend beyond its established role in colorectal cancer:

• Cancer immunotherapy and chaperone biology:

  • ST13's role in the HSP70/HSP90 chaperone system may influence antigen presentation

  • Research could explore how ST13 expression affects response to checkpoint inhibitors

  • ST13 antibodies can help characterize chaperone-dependent processes in immune cells

  • Understanding ST13's role in tumor immune microenvironment may identify new therapeutic targets

• Stress response pathways in neurodegenerative diseases:

  • The HSP70/HSP90 system plays a crucial role in protein folding and degradation

  • ST13 antibodies could help investigate chaperone dysfunction in Alzheimer's and Parkinson's diseases

  • Potential links between ST13 expression and protein aggregation could be explored

  • ST13's role in neuronal stress response might offer insights into disease mechanisms

• Drug resistance mechanisms:

  • HSP90 inhibitors are being developed as cancer therapeutics

  • ST13 antibodies can help study how co-chaperone expression affects drug sensitivity

  • Expression patterns might predict response to HSP90-targeted therapies

  • Understanding the dynamic interactions in the chaperone complex may improve drug design

• Hormone receptor signaling beyond glucocorticoid receptors:

  • ST13's established role in glucocorticoid receptor assembly suggests potential involvement with other nuclear receptors

  • ST13 antibodies can help investigate co-chaperone requirements for estrogen, androgen, or progesterone receptor function

  • This research may identify new therapeutic approaches for hormone-dependent cancers

• Tissue-specific roles in development and differentiation:

  • ST13 expression varies across tissues, suggesting specialized functions

  • Developmental studies using ST13 antibodies could reveal stage-specific roles

  • Understanding tissue-specific co-chaperone requirements may explain differential sensitivity to proteotoxic stress

• Systems biology approaches to chaperone networks:

  • ST13 antibodies enable protein-protein interaction studies

  • Mapping the dynamic chaperone interactome under different conditions

  • Integration with other -omics data to build comprehensive models of cellular stress response