PROK1 Antibody

What experimental techniques can PROK1 antibodies be reliably used for?

PROK1 antibodies have been validated for multiple applications including:

• Immunohistochemistry (IHC): Effective for detecting PROK1 in human colon cancer tissue, mouse kidney tissue, and mouse liver tissue with recommended dilutions of 1:50-1:500

• Immunofluorescence (IF/ICC): Validated in cells like NIH/3T3 with dilutions of 1:50-1:500

• Flow Cytometry (FC): Intra-cellular applications requiring approximately 0.40 μg per 10^6 cells

• Western Blotting: For protein detection in tissue and cell lysates

• ELISA: For quantitative measurement of PROK1 in serum, plasma, and cell culture supernatants

When performing immunohistochemistry with PROK1 antibodies, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may be used as an alternative .

What are the key differences between the available types of PROK1 antibodies?

Researchers can choose between:

• Polyclonal PROK1 antibodies: Such as rabbit polyclonal antibodies that show reactivity with human, mouse, and rat samples. These are generated using recombinant PROK1 immunogens (e.g., Arg28-Phe105 expressed in E. coli)

• Monoclonal PROK1 antibodies: Including mouse monoclonal antibodies validated for specific applications like Cytometric bead arrays and Indirect ELISA. These offer higher specificity for particular epitopes

• Application-specific formats: Including "conjugation ready" formats free of BSA and azide, designed for applications requiring antibody pairs such as multiplex assays

What tissues and cell types express PROK1, and how can antibodies detect this expression?

PROK1 expression has been detected in:

• Colorectal cancer cell lines (DLD-1, HCT116, LoVo) as demonstrated through immunohistochemical staining

• Corpus luteum during pregnancy and the mid-luteal phase

• Human follicular fluid and follicular cell samples with notable interindividual variations

• Endocrine gland cells, where it was originally identified

For detection methods, immunohistochemical staining protocols typically include:

• Fixation in 4% paraformaldehyde

• Rinsing in phosphate-buffered solution containing 0.3% Triton-X

• Blocking with 3% FBS to prevent non-specific binding

• Overnight incubation with anti-PROK1 antibody at 4°C

How can PROK1 antibodies be used to study cancer progression mechanisms?

PROK1 antibodies have proven valuable in studying cancer mechanisms through:

• Tumor angiogenesis assessment: Anti-PROK1 antibodies have been used to evaluate angiogenic activity in vitro and in vivo. Studies show that culture fluid from colorectal cancer cells increases angiogenesis, while anti-PROK1 antibodies suppress this effect .

• Metastasis model studies: In liver metastasis mouse models, anti-PROK1 antibody administration (500 μg intraperitoneally every three days) significantly reduced the number and size of metastatic lesions compared to control groups .

• Cell proliferation analysis: Researchers utilize Ki67 immunostaining in conjunction with PROK1 antibodies to analyze proliferation in metastatic lesions. Studies demonstrate significant decreases in Ki67-positive cells in anti-PROK1 antibody-treated groups:

  • HCT116: 24.3 vs. 74.3 positive cells per visual field (antibody vs. control)

  • HT29: 39.3 vs. 92.3 positive cells per visual field

  • DLD-1: 11.0 vs. 99.7 positive cells per visual field

• Survival analysis: Kaplan-Meier survival curves demonstrate significantly prolonged survival in mice treated with anti-PROK1 antibodies compared to controls (median survival: 46 vs. 31 days, p=0.00273) .

What are the optimal protocols for using PROK1 antibodies in angiogenesis research?

For angiogenesis research, the following methodological approaches have proven effective:

• Subcutaneous angiogenesis assessment:

  • Mix colorectal cancer cell culture fluid with anti-PROK1 antibody

  • Inject the mixture subcutaneously in mouse models

  • Evaluate blood vessel length and diameter

  • Perform immunohistochemical staining with anti-CD31 antibody to quantify vascular density

• In vitro angiogenesis models:

  • PROK1 antibodies can be used to study capillary-like structure formation by endothelial cells

  • Researchers can assess the impact on angiogenin gene expression

  • Measure VEGFA secretion by tissue exposed to PROK1 antibodies

• Combined targeting approach:

  • Studies show enhanced anti-angiogenic effects when combining anti-PROK1 and anti-VEGF antibodies

  • This combined approach demonstrates stronger suppression of tumor formation and peritumoral angiogenesis than either antibody alone

How can researchers accurately quantify PROK1 levels in biological samples?

For precise quantification of PROK1 in biological samples:

What evidence supports the therapeutic potential of anti-PROK1 antibodies in cancer research?

Several lines of evidence support therapeutic applications:

• Tumor growth inhibition:

  • Anti-PROK1 antibody treatment significantly suppresses subcutaneous tumor formation in mouse models

  • When HCT116 cells were injected, tumor size was reduced from 410 mg (control) to 230 mg (anti-PROK1 antibody treatment)

  • The effect was even stronger when combining anti-PROK1 with anti-VEGF antibodies, reducing tumor size to 50 mg

• Survival benefit in metastatic models:

  • Significant extension of survival time has been demonstrated:

    • HCT116 model: 33 vs. 26 days (p=0.00885)

    • HT29 model: 46 vs. 33 days (p=0.0279)

    • DLD-1 model: 130 vs. 46 days (p=0.00249)

• Mechanism of action studies:

  • Anti-PROK1 antibodies suppress angiogenesis by reducing CD31-positive endothelial cells

  • They inhibit cancer cell proliferation through possible effects on transforming growth factor-β (TGF-β) expression

  • PROK1 antibodies may affect multiple intracellular signaling pathways including MAP kinase

How do anti-PROK1 antibodies compare with other anti-angiogenic therapies?

Key comparative insights include:

• Combination potential:

  • When anti-PROK1 and anti-VEGF antibodies are simultaneously applied, tumor formation and peritumoral angiogenesis are more strongly suppressed than with either antibody alone

  • This suggests complementary mechanisms of action that could overcome resistance to single-agent anti-angiogenic therapy

• Unique advantages:

  • Current molecularly-targeted therapeutic agents often require combination with anticancer agents, while anti-PROK1 antibodies show efficacy as monotherapy in preclinical models

  • Anti-PROK1 antibodies may target both angiogenesis and direct cancer cell proliferation through autocrine mechanisms

• Clinical translation considerations:

  • The dosing regimen of 500 μg (20 mg/kg) every three days has shown efficacy in preclinical models

  • Targeting PROK1 may be particularly relevant for advanced colorectal cancer (stages III and IV), where PROK1 expression is significantly higher

How can researchers validate the specificity of PROK1 antibodies?

Recommended validation approaches include:

• Negative controls:

  • For immunofluorescence analyses, include negative controls without primary antibody to confirm the specificity of staining

  • When performing PCR analysis alongside antibody studies, include controls with/without reverse transcriptase enzyme

• Multiple detection methods:

  • Combine techniques such as immunohistochemistry and Western blotting to confirm expression

  • For cell line studies, first confirm PROK1 expression using PCR before proceeding with antibody-based applications

• Cross-reactivity testing:

  • Test antibodies against multiple species when possible, especially if conducting comparative studies

  • Current commercial antibodies show varying reactivity profiles with some specific to human samples and others reactive with human, mouse, and rat samples

What are the optimal storage conditions for maintaining PROK1 antibody activity?

For maximum stability and activity:

• Storage temperature:

  • Most PROK1 antibodies should be stored at -20°C

  • Some conjugation-ready formats require storage at -80°C

• Buffer considerations:

  • Many commercial PROK1 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Some preparations include 0.05% Proclin-300 or other preservatives

• Handling recommendations:

  • Aliquot antibodies to avoid repeated freeze/thaw cycles

  • For some preparations, the small 20μl size contains 0.1% BSA which helps maintain stability

  • Antibodies should be thawed completely before use and mixed gently to ensure homogeneity

How should researchers determine the optimal dilution for PROK1 antibodies in different applications?

To optimize PROK1 antibody performance:

• Application-specific titration:

  • For IHC and IF/ICC: Starting with the recommended range (1:50-1:500), perform dilution series to determine optimal signal-to-noise ratio

  • For flow cytometry: Test concentrations around the recommended 0.40 μg per 10^6 cells

• Sample-dependent optimization:

  • Different sample types may require adjusted antibody concentrations

  • When working with a new tissue or cell type, perform a dilution series specific to that sample

• Confirmatory approaches:

  • Use multiple antibody clones when possible to confirm staining patterns

  • When designing crucial experiments, compare the performance of different commercially available PROK1 antibodies

How are PROK1 antibodies utilized in reproductive biology research?

PROK1 antibodies have proven valuable in several reproductive research areas:

• Corpus luteum function studies:

  • PROK1 antibodies help identify PROK1's role in regulating progesterone synthesis

  • Immunohistochemistry with PROK1 antibodies reveals localization patterns in luteal tissue

• Embryo implantation prediction:

  • PROK1 levels in follicular fluid (FF) and follicular cell mass (FCM) significantly predict embryo implantation outcomes

  • ELISA techniques using PROK1 antibodies achieve high predictive accuracy (84-88%)

• Cell viability and apoptosis assessment:

  • PROK1 antibodies are used in conjunction with FITC-Annexin V and propidium iodide staining to study how PROK1 affects luteal cell apoptosis and viability

  • Results show PROK1 significantly reduces the percentage of apoptosis and increases cell viability in luteal cells

What methodological approaches are recommended for studying PROK1 in embryo implantation?

For embryo implantation research:

• PROK1 quantification in reproductive fluids:

  • Follicular fluid (FF) PROK1 levels >5.47 ng/follicle predict successful pregnancy with 92% sensitivity and 77% specificity

  • Follicular cell mass (FCM) PROK1 levels >29.05 pg/COC predict successful pregnancy with 83% sensitivity and 92% specificity

• ROC curve analysis:

  • The area under the curve for FF PROK1 levels reached 0.916 (95% CI: 0.813-1.000)

  • The area under the curve for FCM PROK1 levels reached 0.878 (95% CI: 0.717-1.000)

  • Both values indicate excellent discriminatory power (p = 7 × 10^-15 and p = 0.0004, respectively)

• Comparative performance metrics: