PROK1 Antibody, Biotin conjugated

What is Prokineticin-1 (PROK1) and why is it important in research?

Prokineticin-1 (PROK1), also known as endocrine gland-derived vascular endothelial growth factor (EG-VEGF), is a protein that induces proliferation, migration, and fenestration (formation of membrane discontinuities) in capillary endothelial cells derived specifically from endocrine glands. PROK1 has minimal effects on other endothelial and non-endothelial cell types. The gene encoding PROK1 has been mapped to chromosome 1 by the International Radiation Hybrid Mapping Consortium. PROK1 expression is primarily restricted to steroidogenic glands including the ovary, testis, adrenal gland, and placenta .

The protein is particularly significant in research because its expression is induced by hypoxia and often complements vascular endothelial growth factor (VEGF) expression patterns, suggesting that these molecules work together in a coordinated manner. This complementary expression pattern makes PROK1 a valuable target for studying angiogenesis in endocrine tissues and potentially for developing therapeutic approaches for disorders involving abnormal vascularization .

What is the molecular structure and function of PROK1?

PROK1 is a relatively small protein with a calculated molecular weight of approximately 11.7 kDa, though it often appears at around 20 kDa in Western blot analysis due to post-translational modifications. The amino acid sequence of human PROK1 includes: AVITGACERDVQCGAGTCCAISLWLRGLRMCTPLGREGEECHPGSHKVPFFRKRKHHTCPCLPNLLCSRFPDGRYRCSMDLKNINF .

Functionally, PROK1 serves as a tissue-specific angiogenic factor that primarily targets endocrine gland endothelial cells. It promotes essential vascular processes including proliferation, migration, and the formation of fenestrations in capillary endothelial cells. These processes are crucial for proper vascularization and function of endocrine organs. Human PROK1 shares significant sequence homology with other species - approximately 88.4% and 91.9% amino acid identity with mouse and rat PROK1, respectively - indicating its evolutionary conservation and biological importance .

How do biotin-conjugated antibodies enhance detection sensitivity in PROK1 research?

Biotin-conjugated antibodies significantly enhance detection sensitivity in PROK1 research through multiple mechanisms. The biotin-streptavidin system provides one of the strongest non-covalent biological interactions known (Kd ≈ 10^-15 M), which creates a robust foundation for signal amplification. This high-affinity interaction allows researchers to develop more sensitive detection methods for PROK1, especially in tissues or samples where expression levels may be low .

In practical applications like sandwich ELISA, a biotin-conjugated anti-PROK1 antibody can be used as the detection antibody while a non-conjugated antibody serves as the capture antibody. This arrangement allows for subsequent addition of streptavidin-conjugated reporter molecules (enzymes, fluorophores, etc.), creating multiple layers of signal amplification. For instance, when using a biotin-conjugated PROK1 antibody in sandwich ELISA at concentrations of 0.25-1.0 μg/mL, researchers can detect as little as 0.2-0.4 ng/well of recombinant human PROK1, demonstrating the high sensitivity achievable with this approach .

What are the optimal conditions for using biotin-conjugated PROK1 antibodies in Western blot applications?

When designing Western blot experiments with biotin-conjugated PROK1 antibodies, researchers should optimize several critical parameters to achieve specific and sensitive detection. Based on empirical data from validation studies, the following protocol has demonstrated reliable results:

For optimal results, protein samples should be transferred to a nitrocellulose membrane at approximately 150 mA for 50-90 minutes. Blocking should be performed with 5% non-fat milk in TBS for 1.5 hours at room temperature. Following incubation with the biotin-conjugated primary antibody overnight at 4°C, thorough washing with TBS-0.1% Tween (3 times, 5 minutes each) is essential before adding streptavidin-HRP. Signal development can be accomplished using enhanced chemiluminescence detection systems .

How should sandwich ELISA be optimized when using biotin-conjugated PROK1 antibodies?

Optimizing sandwich ELISA with biotin-conjugated PROK1 antibodies requires careful consideration of antibody pairing, concentrations, and detection systems. The following methodology has been validated for high sensitivity and specificity:

• Capture Antibody Selection: Use a non-biotinylated anti-PROK1 antibody (e.g., PeproTech Polyclonal Anti-Human EG-VEGF/500-P188G) at 1-2 μg/mL in coating buffer. Coat plates overnight at 4°C.

• Sample Preparation: Prepare standards using recombinant human PROK1 in a concentration range of 0-10 ng/mL. Dilute test samples appropriately in assay buffer containing 0.1% BSA.

• Detection Antibody Application: Apply biotin-conjugated anti-PROK1 antibody at 0.25-1.0 μg/mL (100 μL/well) and incubate for 2 hours at room temperature.

• Signal Development: Use streptavidin-conjugated HRP followed by appropriate substrate. For colorimetric detection, TMB substrate followed by stop solution is recommended.

This optimized protocol enables detection of PROK1 with a sensitivity threshold of 0.2-0.4 ng/well. For quantitative analysis, construct a standard curve using four-parameter logistic regression for accurate interpolation of unknown sample concentrations .

What considerations are important when selecting between polyclonal and monoclonal biotin-conjugated PROK1 antibodies?

The choice between polyclonal and monoclonal biotin-conjugated PROK1 antibodies should be guided by experimental requirements and the specific research questions being addressed:

For studies requiring detection of PROK1 in complex biological samples like serum or tissue homogenates, polyclonal antibodies often provide better sensitivity due to their recognition of multiple epitopes. This is particularly advantageous when studying tissues with low PROK1 expression levels or when protein conformation may be altered. For instance, biotin-conjugated polyclonal PROK1 antibodies have been successfully used to detect native PROK1 in hypoxic endocrine tissues .

In contrast, monoclonal antibodies offer superior specificity and consistency between experiments, making them ideal for longitudinal studies or when comparing PROK1 expression across different experimental conditions. When absolute specificity is critical, such as distinguishing between PROK1 and the closely related PROK2, monoclonal antibodies should be preferred despite their potentially lower signal strength .

How can researchers verify the specificity of biotin-conjugated PROK1 antibodies?

Verifying the specificity of biotin-conjugated PROK1 antibodies is crucial for experimental reliability. Researchers should implement a comprehensive validation strategy that includes the following methodological approaches:

• Positive and Negative Controls: Test the antibody against cell lines with known PROK1 expression profiles. For example, MCF-7 cells have been validated as a positive control for PROK1 expression, showing the expected 20 kDa band in Western blot analysis. Include negative controls such as cell lines where PROK1 is not expressed or PROK1-knockout models .

• Peptide Competition Assay: Pre-incubate the biotin-conjugated PROK1 antibody with excess purified recombinant PROK1 protein before application in your assay. Specific binding should be significantly reduced or eliminated. The recombinant protein should correspond to the immunogen used to generate the antibody, such as E. coli-derived human Prokineticin 1 (Position: A20-F105) .

• Cross-Reactivity Assessment: Test against related proteins, particularly PROK2, which shares structural similarities with PROK1. High-quality PROK1 antibodies should show minimal or no cross-reactivity with other proteins in the prokineticin family. Manufacturers often provide cross-reactivity data that should be reviewed before antibody selection .

• Multiple Detection Methods: Confirm findings using at least two independent detection methods (e.g., Western blot and ELISA) to rule out method-specific artifacts. This multi-platform validation approach substantially increases confidence in antibody specificity .

What are common sources of high background when using biotin-conjugated antibodies and how can they be mitigated?

High background is a frequent challenge when working with biotin-conjugated antibodies, including those targeting PROK1. The following table outlines common sources of background and provides methodological solutions:

For biotin-conjugated PROK1 antibodies specifically, researchers should be aware that steroidogenic tissues (primary targets for PROK1 studies) often contain high levels of endogenous biotin. In these cases, implementing a biotin blocking step is critical for reducing background. Additionally, when working with the PeproTech biotin-conjugated PROK1 antibody, a working concentration of 0.1-0.5 μg/mL for Western blot has been empirically determined to provide the optimal balance between specific signal and background minimization .

What factors might affect the stability of biotin-conjugated PROK1 antibodies and how should they be stored?

Proper storage and handling of biotin-conjugated PROK1 antibodies is essential for maintaining their activity and specificity. Several factors can compromise stability, and researchers should implement specific measures to preserve antibody integrity:

Based on validated protocols, lyophilized biotin-conjugated PROK1 antibodies can be stored at -20°C for up to one year from the date of receipt. After reconstitution, they retain activity for approximately one month when stored at 4°C. For longer storage after reconstitution, aliquoting and freezing at -20°C is recommended, where the antibody remains stable for up to six months. When storing reconstituted PROK1 antibodies, standard buffers containing 5 mg/mL BSA, 0.9 mg/mL NaCl, 0.2 mg/mL Na2HPO4, and 0.05 mg/mL NaN3 have been shown to enhance stability .

How can biotin-conjugated PROK1 antibodies be utilized in studying hypoxia-induced expression in steroidogenic tissues?

Biotin-conjugated PROK1 antibodies offer powerful tools for investigating hypoxia-induced PROK1 expression in steroidogenic tissues. The following methodological approach leverages the high sensitivity of biotin-conjugated antibodies to study this important biological process:

• Experimental Design: Create an in vitro hypoxia model using steroidogenic cell lines (e.g., H295R adrenocortical cells or granulosa cells) exposed to controlled hypoxic conditions (1-3% O2) for varying durations (6, 12, 24, 48 hours). Include normoxic controls (21% O2) for comparison.

• Quantitative Analysis: Employ Western blot using biotin-conjugated PROK1 antibodies (0.1-0.2 μg/mL) to quantify protein expression changes. For enhanced sensitivity, particularly with precious tissue samples, use sandwich ELISA with biotin-conjugated detection antibodies at 0.25-1.0 μg/mL concentration.

• Co-localization Studies: Perform double immunofluorescence staining with biotin-conjugated PROK1 antibodies and antibodies against hypoxia markers (e.g., HIF-1α) to demonstrate spatial relationships between hypoxic regions and PROK1 expression.

• Functional Correlation: Correlate PROK1 expression with angiogenic parameters (e.g., endothelial cell proliferation or microvascular density) to establish functional relevance of hypoxia-induced PROK1 upregulation.

This methodological framework allows researchers to comprehensively study the relationship between hypoxia and PROK1 expression. The biotin-conjugated antibodies provide enhanced sensitivity critical for detecting potentially subtle changes in PROK1 expression patterns, particularly in early response to hypoxic conditions .

What methodological approaches can be used to investigate PROK1 and VEGF co-expression patterns using biotin-conjugated antibodies?

Investigating the complementary expression patterns of PROK1 and VEGF requires sophisticated methodological approaches that take advantage of the signal amplification properties of biotin-conjugated antibodies:

• Sequential Double Immunohistochemistry:

  • First round: Stain for VEGF using conventional (non-biotin) detection systems

  • Intermediate step: Block residual antibodies with glycine-SDS buffer (pH 2.0)

  • Second round: Stain for PROK1 using biotin-conjugated antibodies and streptavidin-conjugated fluorophores or enzymes

  • Analysis: Quantify relative expression levels and spatial distribution patterns

• Multiplex Immunofluorescence Protocol:

  • Primary antibodies: Anti-VEGF (mouse origin) and anti-PROK1 (rabbit origin)

  • Secondary detection: Standard fluorophore for VEGF and biotin-conjugated secondary for PROK1

  • Signal development: Add streptavidin-conjugated fluorophore (with distinct emission spectrum)

  • Counterstain: DAPI for nuclear visualization

  • Analysis: Confocal microscopy with spectral unmixing to distinguish overlapping signals

• Quantitative Co-expression Analysis:

This combined approach allows researchers to quantitatively assess the degree of complementary expression versus co-expression in different steroidogenic tissues under various physiological or pathological conditions. The enhanced sensitivity of biotin-conjugated PROK1 antibodies is particularly advantageous when examining tissues with naturally low PROK1 expression levels .

How can researchers optimize biotinylation of their own PROK1 antibodies for customized applications?

Researchers seeking to biotinylate their own PROK1 antibodies for specialized applications can implement the following optimized protocol based on validated methods:

• Antibody Preparation:

  • Ensure antibody concentration is between 0.5-5.0 mg/mL

  • Buffer exchange to remove incompatible components (e.g., glycerol >1%, primary amines, carriers >0.1%)

  • For antibodies in BSA or other carriers, select a labeling protocol that tolerates these additives

• Biotinylation Procedure Using Mix-n-Stain™ Technology:

  • For 5-20 μg antibody: Use micro-scale kit

  • For 20-50 μg antibody: Use mid-scale kit

  • For 50-100 μg antibody: Use standard-scale kit

  • Add 10X Mix-n-Stain™ Reaction Buffer (1/10 volume)

  • Transfer to vial containing lyophilized biotin reagent

  • Incubate for exactly 15 minutes at room temperature

  • Add storage buffer to quench reaction

  • No purification required - 100% recovery of antibody

• Quality Control Assessments:

  • Determine protein concentration by absorbance at 280 nm

  • Calculate biotin:protein ratio using HABA assay or fluorescent biotin quantification kit

  • Test activity using standard applications (Western blot, ELISA) against known positive controls

  • Compare performance to commercial biotin-conjugated PROK1 antibodies

This approach offers several advantages over traditional biotinylation methods, including minimal hands-on time (less than 30 seconds), rapid completion (15 minutes total), elimination of purification steps, and compatibility with antibodies containing BSA, gelatin, or ascites fluid. Additionally, this method is particularly suitable for precious custom-made PROK1 antibodies available in limited quantities .

How can biotin-conjugated PROK1 antibodies be applied in reproductive biology research?

Biotin-conjugated PROK1 antibodies offer valuable tools for investigating critical processes in reproductive biology, particularly given PROK1's significant role in ovarian, testicular, and placental function. The following methodological approaches demonstrate how these antibodies can advance reproductive research:

• Follicular Development Studies:

  • Immunohistochemistry Protocol: Use biotin-conjugated PROK1 antibodies (0.5-1.0 μg/mL) on ovarian sections with antigen retrieval (citrate buffer, pH 6.0)

  • Quantification Method: Image analysis software to measure PROK1 expression across follicular stages

  • Correlation Analysis: Relate PROK1 expression to follicular vascularity using CD31 co-staining

Researchers have demonstrated that PROK1 expression fluctuates predictably during follicular development, with expression patterns that correlate with follicular vascularity but differ from conventional angiogenic factors like VEGF. This suggests a complementary role in regulating ovarian angiogenesis .

• Placental Development and Pathology:

  • Sandwich ELISA Application: Measure PROK1 in maternal serum using capture antibody (1-2 μg/mL) and biotin-conjugated detection antibody (0.25-1.0 μg/mL)

  • Western Blot Protocol: Analyze placental tissue lysates (30 μg/lane) using biotin-conjugated antibodies at 0.1-0.2 μg/mL

  • Comparative Analysis: Examine expression differences between normal pregnancies and those with complications (e.g., preeclampsia, intrauterine growth restriction)

Studies utilizing these methods have revealed that placental PROK1 expression is significantly altered in pregnancy complications characterized by abnormal placentation. The enhanced sensitivity of biotin-conjugated antibodies allows for reliable detection of PROK1 in maternal circulation, potentially offering a biomarker for monitoring placental health .

What methodological considerations are important when using biotin-conjugated PROK1 antibodies in multiplex assays?

Incorporating biotin-conjugated PROK1 antibodies into multiplex assays requires careful methodological considerations to ensure specificity, sensitivity, and minimal cross-interference:

• Antibody Cross-Reactivity Assessment:

  • Test each antibody individually against all target proteins in the multiplex panel

  • Confirm that biotin-conjugated PROK1 antibodies do not cross-react with other proteins in the multiplex panel

  • Validate specificity using recombinant proteins and knockout/knockdown controls

• Signal Optimization Protocol:

• Multiplex-Specific Controls:

  • Single-analyte positive controls (recombinant PROK1 at known concentrations)

  • Multiplexed positive controls (mixture of all target proteins)

  • Absorption controls (pre-incubation with target antigens to verify specificity)

  • Isotype controls for each species of primary antibody

When implementing these methodological approaches, researchers should be particularly attentive to potential avidin-biotin interference when multiple biotin-conjugated antibodies are used in the same assay. Careful optimization of streptavidin concentration and washing steps is essential to minimize non-specific signals while maintaining sensitivity for PROK1 detection .

How can researchers evaluate the functional impact of PROK1 using biotin-conjugated antibodies in neutralization assays?

Biotin-conjugated PROK1 antibodies can be strategically employed in neutralization assays to evaluate the functional significance of PROK1 in biological processes. The following methodological framework outlines an approach for such studies:

• Antibody Preparation for Neutralization:

  • Select biotin-conjugated polyclonal PROK1 antibodies that recognize functional epitopes

  • Verify epitope specificity using peptide mapping techniques

  • Create F(ab')2 fragments if Fc-mediated effects are a concern

  • Pre-test neutralizing capacity using recombinant PROK1 in a receptor binding assay

• In Vitro Neutralization Protocol:

  • Culture System: Primary endothelial cells derived from endocrine glands

  • Pre-incubation Step: Mix recombinant PROK1 (10-50 ng/mL) with biotin-conjugated antibodies (0.1-10 μg/mL) for 1 hour at 37°C

  • Functional Readouts: Measure proliferation (MTT assay), migration (scratch assay), or tube formation (Matrigel assay)

  • Controls: Include non-biotinylated antibodies to assess if biotinylation affects neutralizing capacity

• Neutralization Efficiency Assessment:

This methodological approach not only confirms the specificity of the biotin-conjugated PROK1 antibodies but also provides valuable insights into the functional significance of PROK1 in endocrine gland angiogenesis. The data from such neutralization studies can help define the concentration-dependent effects of PROK1 and its relative importance compared to other angiogenic factors like VEGF .