PROK1 Antibody, HRP conjugated

What is PROK1 and why is it significant in biological research?

Prokineticin-1 (PROK1), also known as endocrine gland-derived vascular endothelial growth factor (EG-VEGF), is a secreted protein with pleiotropic functions in human and animal tissues. The protein's N-terminal amino acid sequence, AVITGA, is completely conserved and essential to its biological activity . PROK1 plays significant roles in early pregnancy, particularly in endometrial processes during implantation. The protein interacts with its receptor, prokineticin receptor 1 (PROKR1), to regulate the expression of multiple genes critical for successful implantation and early pregnancy maintenance . Research has shown that PROK1 localizes to the syncytiotrophoblast and cytotrophoblast layers in placental tissue, indicating its importance in pregnancy-related processes . Due to its involvement in crucial biological processes, PROK1 has become a valuable target for immunological detection methods in reproductive biology and endocrinology research.

What are the key characteristics of commercially available PROK1 antibodies?

Commercial PROK1 antibodies are available in various forms with specific characteristics optimized for research applications. Typically, these antibodies are developed in rabbit hosts and are available in both non-conjugated and HRP-conjugated formats . The antibodies are commonly polyclonal in nature, targeting multiple epitopes of the PROK1 protein . High-quality PROK1 antibodies, such as those designated as "Picoband," are characterized by superior quality, high affinity, and strong signals with minimal background in Western blot applications . Commercially available antibodies typically target human PROK1, particularly recombinant human Prokineticin-1 protein positions A20-F105, which shares significant amino acid sequence identity with mouse (88.4%) and rat (91.9%) Prokineticin-1 . These antibodies are commonly supplied in lyophilized form or liquid formulations containing preservatives and stabilizers such as BSA, NaCl, Na₂HPO₄, and glycerol to maintain antibody integrity during storage .

What is HRP conjugation and how does it enhance antibody functionality?

Horseradish peroxidase (HRP) conjugation is a process that covalently binds HRP enzyme molecules to antibodies, creating a powerful tool for detection in various immunoassays. The conjugation enhances antibody functionality by enabling colorimetric, chemiluminescent, or fluorescent detection when appropriate substrates are added . During the conjugation process, proprietary reagents activate directional covalent bonding of HRP to the antibody, allowing for specific and sensitive detection of target antigens . This enhancement is particularly valuable in techniques like ELISA, Western blotting, and immunohistochemistry, where signal amplification is necessary for detecting low-abundance proteins. The HRP enzyme catalyzes the oxidation of substrates such as TMB (3,3',5,5'-tetramethylbenzidine) in the presence of hydrogen peroxide, resulting in colored or luminescent products that can be measured quantitatively . The conjugation process is designed to maintain antibody specificity while adding the enzymatic capability of HRP, thereby creating a bifunctional molecule that both recognizes the target antigen and generates detectable signals when appropriate detection reagents are applied.

What applications are PROK1 antibody, HRP conjugated suitable for?

PROK1 antibody, HRP conjugated is primarily designed for enzyme-linked immunosorbent assay (ELISA) applications , making it ideal for quantitative determination of PROK1 in various biological samples. The HRP conjugation provides direct detection capability without requiring secondary antibodies, streamlining experimental protocols and potentially reducing background signals. While ELISA is the primary tested application, HRP-conjugated antibodies can theoretically be employed in other techniques that utilize peroxidase-based detection systems, including Western blotting and immunohistochemistry, though specific validation would be necessary . For Western blot applications, high-quality PROK1 antibodies can detect the protein at its observed molecular weight of approximately 20 kDa, which differs from its calculated molecular weight of 11.7 kDa, possibly due to post-translational modifications . In research contexts, PROK1 antibodies have been successfully employed to investigate PROK1 expression in decidual tissue during early pregnancy and to examine its role in regulating expression of implantation-related genes . These applications make HRP-conjugated PROK1 antibodies valuable tools for reproductive biology, endocrinology, and developmental research.

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

Proper storage and handling of PROK1 antibodies, particularly HRP-conjugated variants, are critical for maintaining optimal activity and extending shelf life. Upon receipt, store the antibody at -20°C or -80°C to maintain protein stability and enzymatic activity of the HRP conjugate . It is essential to avoid repeated freeze-thaw cycles as they can significantly degrade both the antibody and the conjugated HRP enzyme . For lyophilized antibody formats, reconstitution should be performed according to manufacturer specifications, typically using distilled water to achieve the recommended concentration (e.g., adding 0.2 ml of distilled water to yield a concentration of 500 μg/ml) . After reconstitution, the antibody can be stored at 4°C for up to one month . For longer-term storage after reconstitution, aliquot the antibody into smaller volumes to minimize freeze-thaw cycles and store at -20°C for up to six months . The storage buffer typically contains stabilizers such as glycerol (50%) and PBS (0.01M, pH 7.4) along with preservatives like Proclin 300 (0.03%) . When working with HRP-conjugated antibodies, it is critical to avoid buffers containing sodium azide, as it is an irreversible inhibitor of HRP and will compromise the activity of the conjugate .

How should PROK1 antibody dilutions be optimized for different experimental applications?

Optimization of PROK1 antibody dilutions is essential for achieving optimal signal-to-noise ratios in various experimental applications. For ELISA applications using HRP-conjugated PROK1 antibodies, begin with the manufacturer's recommended dilution range, typically 1:1000 to 1:5000, and perform a dilution series to determine the optimal concentration for your specific sample type and assay conditions . For Western blotting applications with non-conjugated PROK1 antibodies, optimization typically involves testing dilutions ranging from 1:500 to 1:2000, followed by detection with an appropriate HRP-conjugated secondary antibody . When optimizing antibody dilutions, consider the abundance of the target protein in your sample; rare proteins may require higher antibody concentrations, while abundant proteins may yield cleaner results with more diluted antibody preparations. The dilution buffer should be compatible with both the antibody and the HRP conjugate, ideally consisting of a protein-containing buffer (e.g., 1-5% BSA or non-fat dry milk in PBS or TBS) to minimize non-specific binding . For each new lot of antibody or when changing experimental conditions (e.g., sample type, detection method), perform a new optimization to ensure consistent and reliable results. Document your optimization experiments thoroughly, including antibody dilutions, incubation times and temperatures, washing conditions, and detection parameters for reproducibility across experiments.

What buffers and reagents are compatible with HRP-conjugated antibodies?

The selection of compatible buffers and reagents is crucial when working with HRP-conjugated antibodies to maintain both antibody binding capacity and enzymatic activity. For antibody dilution and incubation, 10-50 mM amine-free buffers such as HEPES, MES, MOPS, and phosphate with pH ranging from 6.5 to 8.5 are recommended . While moderate concentrations of Tris buffer (<20 mM) may be tolerated, it is important to avoid buffers containing nucleophilic components such as primary amines and thiols (e.g., thiomersal/thimerosal) as they may react with HRP conjugation chemicals . Common non-buffering salts, sugars, and EDTA generally have minimal effects on HRP activity and antibody function . Significantly, sodium azide must be strictly avoided with HRP-conjugated antibodies as it irreversibly inhibits HRP enzymatic activity, rendering the conjugate non-functional . For blocking and washing steps, PBS or TBS containing 0.05-0.1% Tween-20 and protein blockers such as BSA or non-fat dry milk (1-5%) are typically compatible, though some milk preparations may contain endogenous peroxidase activity that could increase background. For substrate selection, common HRP substrates include TMB, DAB (3,3'-diaminobenzidine), and enhanced chemiluminescence (ECL) reagents, which are generally compatible with properly diluted HRP-conjugated antibodies in appropriate buffer systems.

How can PROK1 antibody, HRP conjugated be utilized in a dual-immunofluorescence protocol?

Utilizing PROK1 antibody, HRP conjugated in dual-immunofluorescence protocols requires strategic planning to leverage the HRP component while allowing simultaneous detection of a second target. First, prepare tissue sections or cell samples through standard fixation and antigen retrieval procedures. For paraffin-embedded sections, dewax and rehydrate in graded ethanol as described in established protocols . Block nonspecific binding sites using 5% normal serum from a species different from those in which your primary antibodies were raised . Apply the HRP-conjugated PROK1 antibody at optimized dilution (typically 1:250 to 1:1000) along with a non-conjugated primary antibody against your second target protein (e.g., COX-2, CD31, or CD56) and incubate overnight at 4°C . After thorough washing with PBS containing 0.1% Tween-20, detect the HRP-conjugated PROK1 antibody using tyramide signal amplification (TSA), which converts the HRP enzymatic activity into a stable fluorescent signal. Subsequently, apply an appropriate fluorescently-labeled secondary antibody to detect the non-conjugated primary antibody targeting your second protein. After final washing steps, counterstain nuclei with DAPI and mount slides with an anti-fade mounting medium. This approach enables visualization of PROK1 colocalization with other proteins of interest, such as COX-2 in endometrial cells or CD56 in natural killer cells, providing valuable insights into the functional relationships between PROK1 and other proteins in biological contexts .

What controls should be included when using PROK1 antibody, HRP conjugated in ELISA?

When designing ELISA experiments using PROK1 antibody, HRP conjugated, incorporating appropriate controls is essential for result validation and troubleshooting. A comprehensive control strategy should include positive controls using recombinant human Prokineticin-1 protein (positions A20-F105) at known concentrations to establish assay performance and standard curves . Negative controls should include samples known to lack PROK1 expression or samples from PROK1 knockout models if available. To account for non-specific binding, include isotype controls using non-specific rabbit IgG, HRP-conjugated at the same concentration as your PROK1 antibody . Background controls consisting of all reagents except the primary antibody will help determine signal contribution from non-specific binding of detection reagents. For validating antibody specificity, include blocking peptide controls where the HRP-conjugated PROK1 antibody is pre-incubated with excess PROK1 immunogen peptide before application to the assay . Serial dilution controls of your sample will help establish the linear range of detection and optimal sample concentration. Inter-assay controls consisting of the same sample analyzed across multiple plates or experiments will allow assessment of reproducibility. Include these controls on each ELISA plate and analyze them alongside your experimental samples to ensure valid interpretation of results and to troubleshoot any unexpected outcomes in your PROK1 detection assays.

How does PROK1-PROKR1 signaling influence gene expression, and how can this be studied?

PROK1-PROKR1 signaling orchestrates a complex gene expression program crucial for implantation and early pregnancy. Gene microarray analysis of Ishikawa PROKR1 cells treated with 40 nm PROK1 for 8 hours identified 49 differentially regulated genes, including cyclooxygenase (COX)-2, leukemia inhibitory factor, IL-6, IL-8, and IL-11, which are known to be regulated in the endometrium during implantation and early pregnancy . This signaling cascade operates through the Gq-phospholipase C-β-cSrc-epidermal growth factor receptor-MAPK/ERK kinase pathway, ultimately influencing the expression of implantation-related genes . To study this signaling mechanism and its effects on gene expression, researchers can employ a multi-faceted approach: Begin with cell models expressing PROKR1 (either endogenous or recombinant) treated with PROK1 at various concentrations and time points. Use quantitative RT-PCR to validate expression changes of key target genes identified through microarray analysis. Western blotting with specific antibodies can confirm protein-level changes and activation of pathway components. Functional assays such as prostaglandin synthesis measurement (PGE2 and PGF2α) can assess downstream effects of pathway activation, as PROK1 treatment increased COX-2 expression and prostaglandin production in previous studies . Pathway inhibitor studies using specific blockers for different components of the signaling cascade can elucidate the precise mechanisms involved. For in vivo relevance, immunohistochemistry with PROK1 and PROKR1 antibodies on decidual tissue sections can confirm expression patterns during early pregnancy . This comprehensive approach provides mechanistic insights into how PROK1-PROKR1 signaling influences the endometrial environment during crucial reproductive processes.

How can PROK1 antibody, HRP conjugated be used to investigate the role of PROK1 in early pregnancy complications?

Investigating the role of PROK1 in early pregnancy complications using HRP-conjugated PROK1 antibodies requires a multi-dimensional approach that leverages the antibody's specificity and detection sensitivity. Begin by collecting decidual and placental tissue samples from both normal pregnancies and those with complications such as recurrent pregnancy loss, preeclampsia, or intrauterine growth restriction, ensuring appropriate ethical approvals and informed consent . Process these tissues for both protein extraction (for quantitative analysis) and histological sectioning (for localization studies). For quantitative assessment, develop an ELISA protocol using the HRP-conjugated PROK1 antibody to measure PROK1 protein levels across sample groups, establishing whether dysregulation occurs in complicated pregnancies . For tissue localization, perform immunohistochemistry on tissue sections using the HRP-conjugated PROK1 antibody to visualize distribution patterns, comparing normal versus complicated pregnancies . Dual immunofluorescence can reveal colocalization of PROK1 with markers of specific cell types such as CD56+ uterine natural killer cells or CD31+ endothelial cells, which may be altered in pregnancy complications . In parallel, assess downstream effects of PROK1 signaling by measuring expression of target genes like COX-2, leukemia inhibitory factor, and interleukins using qPCR and Western blotting . For functional validation, establish primary cell cultures from normal and complicated pregnancy tissues, treating them with recombinant PROK1 to compare signaling responses through assessment of target gene activation and prostaglandin production . This comprehensive approach can reveal whether aberrant PROK1 expression or signaling contributes to the pathophysiology of early pregnancy complications, potentially identifying new therapeutic targets.

What are the technical considerations for performing HRP conjugation to PROK1 antibodies in-house?

Performing in-house HRP conjugation to PROK1 antibodies requires careful attention to technical parameters and optimization steps to ensure functional conjugates. Begin with high-purity antibodies (>95% purity, preferably protein G or protein A purified) in an amine-free buffer at pH 6.5-8.5, as recommended for conjugation reactions . For optimal conjugation, adjust the antibody concentration to 0.5-5.0 mg/ml in a volume of up to 100 μl . Select an appropriate conjugation kit, such as the LYNX Rapid HRP Antibody Conjugation Kit, which contains pre-prepared lyophilized HRP mix, modifier reagent, and quencher reagent . The optimal molar ratio of antibody to HRP should be between 1:1 and 1:4, which for a 100 μg HRP preparation would require 100-400 μg of antibody, considering the molecular weights of typical antibodies (160 kDa) versus HRP (40 kDa) . During conjugation, strictly avoid buffers containing nucleophilic components such as primary amines and thiols, as these can interfere with the conjugation chemistry . Also ensure that sodium azide is completely removed from antibody preparations, as it irreversibly inhibits HRP activity . Follow the conjugation protocol precisely: add the modifier reagent to the antibody preparation (1 μl for every 10 μl of antibody), mix gently, then add this mixture directly to the lyophilized HRP mix . After the recommended incubation period (3 hours at room temperature or overnight), add the quencher reagent (1 μl for every 10 μl of antibody) and allow to stand for 30 minutes before use . Post-conjugation validation should include functional assays comparing the conjugated antibody to a commercial equivalent, assessing both antigen binding capacity and HRP enzymatic activity.

How can variations in observed molecular weight of PROK1 be interpreted in experimental results?

Variations in the observed molecular weight of PROK1 in experimental results require careful interpretation based on knowledge of the protein's biology and experimental considerations. The calculated molecular weight of human PROK1 is approximately 11.7 kDa, yet Western blot analysis often reveals bands at approximately 20 kDa . This discrepancy can be attributed to several biological and technical factors. Post-translational modifications, particularly glycosylation, can significantly increase the apparent molecular weight of PROK1. The human PROK1 protein contains potential N-linked and O-linked glycosylation sites that, when occupied, add substantial mass to the protein . Different tissue sources may express PROK1 variants with tissue-specific post-translational modification patterns, resulting in different observed molecular weights across sample types . Additionally, PROK1 may form stable dimers or complexes with other proteins that are resistant to standard reducing conditions in SDS-PAGE, leading to higher molecular weight bands. From a technical perspective, the type of gel system, running conditions, and molecular weight standards used can all influence the apparent molecular weight. Researchers should validate observed bands through positive controls using recombinant PROK1 protein run alongside experimental samples . For definitive identification of PROK1 isoforms or modified versions, consider additional techniques such as mass spectrometry or 2D gel electrophoresis followed by Western blotting. When unexpected molecular weight variations are observed, researchers should investigate whether these represent physiologically relevant isoforms or modifications that may have functional significance in the biological system under study.

What are common issues encountered when using PROK1 antibody, HRP conjugated, and how can they be resolved?

When working with PROK1 antibody, HRP conjugated, researchers may encounter several common issues that can be systematically addressed through proper troubleshooting. High background signal in ELISA or Western blot can result from insufficient blocking, inadequate washing, or excessive antibody concentration. To resolve this, optimize blocking conditions (try 3-5% BSA or non-fat dry milk in PBS-T), increase washing steps (4-5 washes of 5 minutes each), and test a dilution series of the antibody to determine optimal concentration . Weak or absent signal may occur due to low target protein abundance, degraded antibody, or inhibited HRP activity. Address this by increasing sample concentration, verifying antibody storage conditions, and ensuring no sodium azide or other HRP inhibitors are present in your buffers . Non-specific bands in Western blot can be minimized by optimizing antibody dilution, increasing blocking stringency, and including competitor proteins in the antibody dilution buffer . Cross-reactivity with non-target proteins may occur particularly in complex samples. Validate specificity using recombinant PROK1 protein as a positive control and consider pre-absorption with the immunizing peptide to confirm specificity . Batch-to-batch variability of results can be mitigated by establishing internal controls and standardization procedures for each new lot of antibody. HRP activity loss during storage is another common issue; prevent this by storing the conjugated antibody in aliquots at -20°C or -80°C and avoiding repeated freeze-thaw cycles . For persistent issues, consider alternative detection methods or antibody clones, and always include appropriate positive and negative controls to aid in interpreting unexpected results.

How can researchers validate the specificity of PROK1 antibody, HRP conjugated for their particular application?

Validating the specificity of PROK1 antibody, HRP conjugated for particular applications is essential for ensuring reliable experimental results. Begin with positive controls using recombinant human Prokineticin-1 protein (positions A20-F105) to confirm that the antibody detects the intended target . Complement this with negative controls such as cell lines or tissues known not to express PROK1, or ideally, PROK1 knockout models if available. A critical specificity test involves competitive inhibition using the immunizing peptide; pre-incubate the antibody with excess immunogen peptide before application to your sample – specific signals should be significantly reduced or eliminated . For Western blot applications, examine whether the detected band appears at the expected molecular weight (approximately 20 kDa for PROK1, though post-translational modifications may affect this) . Consider orthogonal validation by comparing results with alternative detection methods, such as mass spectrometry or using a second antibody targeting a different epitope of PROK1. RNA-level validation can provide additional confidence; confirm PROK1 protein expression patterns correlate with mRNA expression detected by RT-PCR or RNA-seq in the same samples. For tissue sections, compare the PROK1 staining pattern with published literature on PROK1 localization, such as its known expression in syncytiotrophoblast and cytotrophoblast layers . Cross-reactivity testing with related proteins, particularly PROK2 which shares structural similarities with PROK1, can further confirm specificity. Document all validation experiments thoroughly, including positive and negative controls, to support the reliability of your results when using the PROK1 antibody, HRP conjugated in your specific research application.

What methodological approaches can optimize detection sensitivity when PROK1 expression is low?

Optimizing detection sensitivity for low PROK1 expression requires methodological refinements across sample preparation, antibody usage, and signal development stages. Begin with sample enrichment techniques such as immunoprecipitation to concentrate PROK1 protein before analysis or employ tissue microdissection to isolate specific cell populations with higher PROK1 expression . For protein extraction, use optimized lysis buffers containing appropriate protease inhibitors to preserve intact PROK1 and prevent degradation during processing. When designing ELISA protocols, employ a sandwich ELISA format using a capture antibody against PROK1 and the HRP-conjugated PROK1 antibody for detection, which can significantly enhance sensitivity compared to direct ELISA formats . Consider extending primary antibody incubation times (overnight at 4°C) and optimizing antibody concentration through careful titration experiments to determine the concentration that provides maximum specific signal with minimal background . Signal amplification systems can dramatically improve sensitivity; options include tyramide signal amplification (TSA) which can enhance HRP-based detection by up to 100-fold, or biotin-streptavidin systems for multi-layered signal enhancement . For Western blotting, use high-sensitivity chemiluminescent substrates specifically designed for low-abundance proteins, and consider longer exposure times when imaging. Reduce background interference by optimizing blocking conditions (test different blocking agents like BSA, casein, or commercial blocking reagents) and implementing more stringent washing protocols. Finally, consider digital imaging systems with wide dynamic range and high sensitivity for signal capture, and employ image analysis software to quantify even faint signals accurately. These methodological refinements, implemented systematically, can significantly enhance detection of low-abundance PROK1 in challenging samples.

Research Applications Table