CST5 Antibody, Biotin conjugated

What is CST5 and what is its biological significance?

CST5 (Cystatin D) functions as an inhibitor of cysteine proteases of the cathepsin family and is directly induced by the vitamin D receptor (VDR) . It plays a significant role in mesenchymal-epithelial transition (MET) in colorectal cancer cells . Research has also identified CST5 as a direct target gene of the p53 tumor suppressor, with expression being upregulated at both mRNA and protein levels after p53 activation . This regulation appears to be direct, as both ectopic and endogenous p53 occupy a conserved binding site in the CST5 gene .

More recently, CST5 has gained attention as a novel early biomarker for traumatic brain injury (TBI). Studies have shown that CST5 can identify patients with severe TBI from other cohorts within the first hour of injury, making it a potentially valuable diagnostic tool in emergency medicine .

What applications can CST5 Antibody, Biotin conjugated be used for?

The CST5 Antibody, Biotin conjugated has been validated for several immunological applications including:

• Enzyme Immunoassays (EIA)

• General immunoassay procedures

• Enzyme-Linked Immunosorbent Assays (ELISA)

This antibody is particularly useful in sandwich ELISA methods, where it serves as the detection antibody that binds to human CST5 protein after it has been captured by a pre-coated antibody on the assay plate . The biotin conjugation allows for subsequent binding of streptavidin-HRP complexes, enabling colorimetric detection through substrate reactions .

What sample types can be analyzed for CST5 detection?

CST5 can be detected in multiple biological samples including:

• Cell culture supernatants

• Serum and plasma (including those prepared with heparin or EDTA)

• Saliva

• Urine

• Tissue homogenates

• Other biological fluids

The versatility in sample compatibility makes CST5 antibodies valuable for various research contexts, from basic cell culture studies to clinical sample analysis.

What is the principle behind sandwich ELISA for CST5 detection?

The sandwich ELISA method for CST5 detection follows these key steps:

• A microtiter plate is pre-coated with an antibody specific to CST5

• Standards or samples containing CST5 are added to the wells

• A biotinylated detection antibody specific for CST5 is added, which binds to any captured CST5

• Avidin conjugated to Horseradish Peroxidase (HRP) is added, which binds to the biotin on the detection antibody

• TMB substrate solution is added, producing a color change in wells containing the complete antibody-antigen complex

• The reaction is stopped with an acidic solution, and absorbance is measured at 450nm

• The concentration of CST5 in samples is determined by comparing to a standard curve

This methodology provides high specificity and sensitivity for CST5 detection, with typical detection ranges between 0.32-20 ng/mL and sensitivity around 0.135 ng/mL for commercial kits .

How should I design appropriate controls for experiments using CST5 Antibody?

When designing experiments with CST5 Antibody, include these essential controls:

Positive Controls:

• Cell lines or tissues known to express CST5, such as colorectal cancer cell lines (e.g., SW480, HCT116) treated with vitamin D3 or after p53 activation

• Recombinant CST5 protein at known concentrations for standard curves

• Samples from conditions known to elevate CST5, such as severe traumatic brain injury specimens

Negative Controls:

• p53-deficient cell variants (e.g., HCT116 p53−/− cells) which show minimal CST5 induction

• Samples where the primary or detection antibody is omitted

• Samples pre-treated with blocking peptides specific to the antibody epitope

When validating antibody specificity, consider using CST5 siRNA knockdown samples as demonstrated in studies where CST5 protein became undetectable after siRNA treatment even with p53 induction .

What are the recommended sample preparation methods for different specimen types?

For Serum:

• Collect blood in serum separator tubes

• Allow samples to clot for two hours at room temperature or overnight at 4°C

• Centrifuge for 20 minutes at approximately 1,000×g

• Assay fresh serum immediately or store aliquots at -20°C or -80°C

• Avoid repeated freeze/thaw cycles

For Plasma:

• Collect using EDTA or heparin as anticoagulant

• Centrifuge samples for 15 minutes at 1,000×g at 2-8°C within 30 minutes of collection

• Remove plasma and assay immediately or store appropriately

For Cell Culture Supernatants:

• Centrifuge to remove particulates

• Assay immediately or aliquot and store at -20°C or -80°C

How can I optimize detection of CST5 in traumatic brain injury research?

CST5 has been identified as a novel early biomarker of traumatic brain injury (TBI), with particular value in identifying severe TBI cases. Research indicates that:

• CST5 levels are significantly increased in severe TBI patients compared to mild TBI and healthy volunteers

• The highest levels of CST5 are detected within the first hour post-injury

• CST5 levels remain elevated (and significantly higher than in mild TBI) at the 4-12 hour time point

• By 48-72 hours, CST5 levels are no longer significantly different from healthy volunteers

For optimal detection in TBI research:

• Collect samples as early as possible post-injury, ideally within the first hour

• Include appropriate controls (healthy volunteers, mild TBI patients)

• Consider a longitudinal sampling approach (e.g., <1 hr, 4-12 hr, 48-72 hr timepoints)

• Use high-sensitivity ELISA kits with detection ranges appropriate for the expected concentration changes

• Consider multiplexing CST5 with other early TBI biomarkers such as AXIN1 and TRAIL for comprehensive evaluation

What are the considerations when studying CST5 in relation to p53 and vitamin D signaling?

When designing experiments to investigate the relationship between p53, vitamin D receptor (VDR), and CST5 expression, consider:

• Temporal dynamics: CST5 induction by p53 follows similar kinetics to other p53 targets like p21. Detectable induction occurs at 24 hours and remains elevated at 72 hours post-activation .

• Activation methods: Multiple approaches can be used to activate p53-dependent CST5 expression:

  • Conditional expression systems (e.g., doxycycline-inducible p53)

  • Small-molecule MDM2 inhibitors (e.g., nutlin-3a)

  • DNA-damaging agents (e.g., etoposide)

• Cell systems: Use matched p53 wild-type and p53-deficient cells (e.g., HCT116 p53+/+ and HCT116 p53−/−) to confirm p53 dependence .

• Functional analysis: To study CST5's role in p53-mediated cellular processes:

  • Use siRNA approaches to silence CST5 expression

  • Assess the impact on downstream targets (e.g., SNAIL protein)

  • Evaluate functional outcomes such as cellular migration using scratch/wound-closure assays and Boyden-chamber assays

What troubleshooting strategies should I implement when working with CST5 Antibody in Western blots?

When troubleshooting Western blot experiments using CST5 antibodies, consider these strategies:

• Appropriate gel selection: Select the right gel percentage based on CST5's molecular weight. For optimal resolution of proteins in CST5's molecular weight range, consider:

  Gel Type	Protein Molecular Weight
  3-8% Tris-Acetate	> 200 kDa
  4-20% Tris-Glycine	10-200 kDa
  10% Tris-Glycine	30-100 kDa
  12% Tris-Glycine	20-80 kDa
  15% Tris-Glycine	10-50 kDa

• Post-translational modifications: Since CST5 can be regulated by p53 and VDR, consider whether specific treatments might be needed to induce the post-translational modification state you're investigating .

• Antibody validation: Ensure you're working with validated antibody by checking:

  • Specificity through siRNA knockdown experiments

  • Expected molecular weight of the target

  • Appropriate positive controls (p53-activated colorectal cancer cells)

• Signal optimization: If signal is weak, consider:

  • Increasing protein loading

  • Extended antibody incubation times

  • Enhanced chemiluminescence detection systems

  • Signal amplification methods compatible with biotin-conjugated antibodies

How can CST5 detection be incorporated into studies of epithelial-mesenchymal transition?

CST5 has been shown to mediate mesenchymal-epithelial transition (MET) in colorectal cancer cells . When designing studies investigating this role:

• Consider the regulatory relationship between p53, CST5, and epithelial-mesenchymal markers such as SNAIL. Research indicates that CST5 silencing reduces p53-mediated repression of SNAIL protein .

• Include functional assays that measure cell migration, such as:

  • Scratch/wound-closure assays

  • Boyden-chamber assays

• Design experiments to test both gain-of-function (through ectopic expression) and loss-of-function (through siRNA) approaches to confirm CST5's role in MET.

• Combine CST5 measurements with analysis of other epithelial and mesenchymal markers to comprehensively assess transition states.

What are important considerations for quantitative analysis of CST5 expression?

For accurate quantification of CST5:

• Standard curve preparation: Create a reliable standard curve using recombinant CST5 protein. Commercial ELISA kits typically provide lyophilized standards with concentrations around 20 ng/mL .

• Detection range: Ensure your assay can detect CST5 within the physiological or pathological range of interest. Commercial kits typically offer detection ranges of 0.32-20 ng/mL with sensitivity around 0.135 ng/mL .

• Data analysis: For ELISA results, plot the standard curve using appropriate curve-fitting methods (typically 4-parameter logistic regression) and use this to calculate sample concentrations based on OD values.

• Normalization strategies: When comparing CST5 levels across different samples or conditions:

  • For Western blots, normalize to appropriate loading controls

  • For tissue samples, consider normalization to total protein content

  • For longitudinal studies, establish baseline values for each subject