KISS1 Antibody, HRP conjugated

What is KISS1 and why is it a significant target for antibody-based detection?

KISS1 functions as a metastasis suppressor protein in malignant melanomas and certain breast cancers. It regulates events downstream of cell-matrix adhesion, potentially involving cytoskeletal reorganization. The protein generates a C-terminally amidated peptide called metastin, which serves as the endogenous ligand of the G-protein coupled receptor GPR54 . Activation of this receptor inhibits cell proliferation and migration, which are key characteristics involved in tumor metastasis . Additionally, the hypothalamic KISS1/GPR54 system plays a pivotal role in the central regulation of the gonadotropic axis during puberty and adulthood . This multifunctional nature of KISS1 makes it an important target for research in oncology, reproductive biology, and developmental studies.

What are the recommended applications for KISS1 Antibody, HRP conjugated?

The KISS1 Antibody, HRP conjugated (catalog #bs-0749R-HRP) is validated for multiple research applications including Western Blotting (WB), Enzyme-Linked Immunosorbent Assay (ELISA), and immunohistochemistry on both paraffin-embedded (IHC-P) and frozen sections (IHC-F) . The recommended dilution ranges vary by application: 1:300-5000 for Western blotting and 1:500-1000 for ELISA protocols . This HRP conjugation eliminates the need for secondary antibody incubation, streamlining experimental workflows and potentially reducing background signal in certain applications. The antibody specifically detects the Kisspeptin-10 region of the KISS1 protein, allowing researchers to investigate this physiologically important segment of the molecule .

What species reactivity can I expect with the KISS1 Antibody, HRP conjugated?

The KISS1 Antibody, HRP conjugated demonstrates confirmed reactivity with human, mouse, and rat samples . Additionally, based on sequence homology and epitope conservation, this antibody is predicted to cross-react with KISS1 from cow and sheep samples, though this reactivity may require validation in individual laboratories . When designing experiments with tissue samples from species not listed in the confirmed reactivity panel, researchers should conduct preliminary validation studies to confirm antibody binding and specificity before proceeding with full experimental protocols.

How should KISS1 Antibody, HRP conjugated be stored to maintain optimal activity?

For optimal preservation of activity, KISS1 Antibody, HRP conjugated should be stored at -20°C . The antibody is supplied in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol . To prevent activity loss from repeated freeze-thaw cycles, it is strongly recommended to aliquot the antibody into multiple smaller volumes upon receipt . Each aliquot should be sized appropriately for single-experiment use to minimize the need for repeated thawing of the same vial. This storage strategy helps maintain the structural integrity of the antibody and preserves the activity of the HRP conjugate, which can be sensitive to repeated temperature fluctuations.

How can I optimize Western blot protocols when using KISS1 Antibody, HRP conjugated for detecting low abundance KISS1 in complex tissue samples?

Detecting low abundance KISS1 protein in complex tissue samples requires careful protocol optimization. Begin with sample enrichment through subcellular fractionation, as KISS1 is primarily localized in secretory pathways . For Western blotting, load increased protein (50-100μg) and use a longer transfer time (overnight at 30V) to ensure complete transfer of proteins. Since this antibody specifically targets the Kisspeptin-10 region , be aware that detection sensitivity may vary depending on the processing status of KISS1 in your samples.

When developing the blot, utilize enhanced chemiluminescence substrates optimized for HRP detection with extended exposure times (initial testing with 30 seconds, 2 minutes, and 5 minutes). The recommended dilution range of 1:300-5000 should be tested systematically, starting with higher concentrations (1:300) for low abundance samples. For complex tissues like hypothalamus or placenta where multiple isoforms may be present, consider using gradient gels (4-20%) to achieve better separation of closely sized protein variants.

What are the critical considerations when performing immunohistochemistry with KISS1 Antibody, HRP conjugated on reproductive tissue samples?

When performing immunohistochemistry on reproductive tissues using KISS1 Antibody, HRP conjugated, several tissue-specific considerations must be addressed. First, reproductive tissues often contain endogenous peroxidase activity, which can generate false-positive signals. Implement a robust peroxidase blocking step using 3% hydrogen peroxide in methanol for 15-20 minutes at room temperature . Additionally, reproductive tissues frequently exhibit high background due to non-specific binding; use 5% normal serum from the same species as your secondary antibody (though not needed with direct HRP conjugates) plus 1% BSA in your blocking buffer.

For antigen retrieval, citrate buffer (pH 6.0) heat-induced epitope retrieval tends to work well for KISS1 detection in reproductive tissues . When examining hypothalamic tissues, consider thinner sections (4μm rather than standard 5-6μm) to better visualize KISS1-expressing neurons. For placental tissue, where KISS1 expression varies by trimester and specific cell populations, precisely document the gestational age and sampling location. Compare your staining patterns with the established KISS1 expression in specific nuclei of the hypothalamus or trophoblast populations in placenta to confirm specificity of your results.

How can I validate the specificity of KISS1 Antibody, HRP conjugated in my experimental system?

Rigorous validation of antibody specificity is essential for reliable experimental outcomes. For KISS1 Antibody, HRP conjugated, implement a multi-faceted validation approach. Begin with positive and negative control tissues: hypothalamic arcuate nucleus and placenta serve as good positive controls, while tissues known to lack KISS1 expression (such as adult liver) serve as negative controls .

Include a peptide competition assay by pre-incubating the antibody with excess synthetic peptide corresponding to the immunogen (peptide from the 81-145 region of human KISS1) , which should eliminate specific staining. For genetic validation, compare staining between wild-type tissues and those from KISS1 knockout models, if available. Alternatively, use KISS1-silenced cell lines created through siRNA or CRISPR technologies.

Cross-validate your findings using an unconjugated version of the same antibody (bs-0749R) or antibodies targeting different epitopes of KISS1 to confirm staining patterns. When possible, correlate protein detection with mRNA expression data from qPCR or in situ hybridization to provide orthogonal validation of expression patterns. Document these validation steps thoroughly to strengthen the credibility of your experimental findings.

What are the most common causes of false negative results when using KISS1 Antibody, HRP conjugated, and how can they be addressed?

False negative results when using KISS1 Antibody, HRP conjugated can stem from multiple experimental factors. Insufficient antigen retrieval is a primary concern, particularly for formalin-fixed tissues where cross-linking may mask the Kisspeptin-10 epitope . To address this, optimize your antigen retrieval method by testing different buffers (citrate pH 6.0 vs. EDTA pH 9.0) and durations (10-30 minutes).

The HRP conjugate may lose activity over time or with improper storage. Test the activity of your antibody using a known positive control sample . If signal is weak despite good antibody activity, consider signal amplification systems compatible with HRP, such as tyramide signal amplification. For Western blotting applications, ensure complete protein transfer (verify with reversible staining of the membrane) and consider using PVDF membranes which may retain small peptides better than nitrocellulose.

KISS1 is often expressed at low levels and undergoes post-translational processing, resulting in multiple peptide products including Kisspeptin-10, Kisspeptin-14, and Kisspeptin-54 . Ensure your extraction method preserves these peptides and use protease inhibitors specifically designed to prevent degradation of small peptides. Finally, because KISS1 expression is highly regulated by physiological state, particularly in reproductive tissues, consider the possibility that your experimental conditions (estrus cycle stage, developmental stage, pathological state) may naturally result in low KISS1 expression.

How can I determine the optimal antibody concentration for my specific experimental conditions?

Determining the optimal concentration of KISS1 Antibody, HRP conjugated requires systematic titration across the recommended dilution ranges for each application: 1:300-5000 for Western blotting and 1:500-1000 for ELISA . Begin your optimization with a positive control sample of known KISS1 expression, ideally from the same species as your experimental samples to account for potential cross-species variation in affinity.

For Western blotting, prepare a dilution series (e.g., 1:300, 1:1000, 1:3000, 1:5000) and process identical membrane strips with equal protein loading. Evaluate the resulting signal-to-noise ratio, looking for the dilution that provides strong specific bands with minimal background. For immunohistochemistry applications, similar serial dilutions should be tested on serial sections of positive control tissue.

An important consideration for HRP-conjugated antibodies is that while higher concentrations may increase sensitivity, they can also increase background staining. If high background persists even at more dilute antibody concentrations, consider implementing additional blocking steps or increasing the concentration of blocking proteins in your buffer. Document your optimization process with images showing the different dilutions tested and the rationale for your final selection to facilitate reproducibility and troubleshooting.

How should I interpret differences in KISS1 detection patterns between different tissue types when using this antibody?

When analyzing KISS1 expression across tissue types, consider that the protein undergoes tissue-specific post-translational processing. In hypothalamic neurons, KISS1 is processed into various kisspeptins (KP-54, KP-14, KP-13, and KP-10), while in placenta and some cancers, processing patterns may differ . Since the antibody detects the Kisspeptin-10 region, differences in band patterns on Western blots or staining intensity in immunohistochemistry may reflect variations in processing rather than total KISS1 expression.

The subcellular localization of KISS1 also varies by tissue type. In hypothalamic neurons, KISS1 is found in secretory vesicles and may show punctate cytoplasmic staining, while in metastatic cancer cells, distribution patterns may differ based on invasion status . Additionally, KISS1 expression is highly regulated by physiological context – in reproductive tissues, expression varies dramatically across the estrous cycle, pregnancy states, and developmental stages.

When comparing tissues, implement quantitative methods such as densitometry for Western blots or digital image analysis for immunohistochemistry to objectively assess differences. Always include appropriate tissue-specific controls and consider complementing antibody-based detection with mRNA analysis to distinguish between transcriptional regulation and post-translational differences.

How can KISS1 Antibody, HRP conjugated be used to investigate the relationship between KISS1 expression and tumor metastasis?

To investigate KISS1's role in tumor metastasis, the HRP-conjugated antibody can be employed in several strategic approaches. Begin with a comprehensive tissue microarray analysis comparing primary tumors, metastatic lesions, and normal tissues to establish expression patterns and correlation with clinical outcomes . The direct HRP conjugation allows for more consistent staining across large sample sets by eliminating secondary antibody variation.

For mechanistic studies, implement dual immunofluorescence (using unconjugated primary antibodies) to co-localize KISS1 with markers of epithelial-mesenchymal transition (E-cadherin, vimentin) or invasion (matrix metalloproteinases). In cell culture models, combine Western blot quantification of KISS1 expression with functional assays such as invasion, migration, and proliferation to correlate expression levels with metastatic behaviors.

Create an experimental matrix comparing KISS1 expression in:

• Non-metastatic versus metastatic cell lines from the same tumor type

• Isogenic cell lines with manipulated KISS1 expression (overexpression or knockdown)

• Primary tumor samples from patients with and without subsequent metastasis

• Different regions within heterogeneous tumors

This approach can reveal whether KISS1 expression correlates inversely with metastatic potential, as would be expected for a metastasis suppressor, and whether this relationship holds across different cancer types and genetic backgrounds.

What considerations are important when using this antibody to study KISS1/GPR54 signaling in reproductive neuroendocrine research?

When studying KISS1/GPR54 signaling in reproductive neuroendocrine systems, several specialized considerations become important. First, KISS1 expression in the hypothalamus is anatomically restricted to specific nuclei (arcuate and anteroventral periventricular nucleus) and is highly responsive to hormonal feedback . Therefore, precisely document the estrous cycle stage, age, and hormonal status of experimental animals, as these factors dramatically influence KISS1 expression.

For co-localization studies examining KISS1 and its receptor GPR54, consider that while KISS1 is produced in kisspeptin neurons, GPR54 is expressed on target cells including GnRH neurons. Sequential immunohistochemistry or fluorescent multiplexing will be necessary to visualize this relationship. When quantifying KISS1-positive cells in the hypothalamus, employ stereological counting methods on serial sections to accurately assess total cell populations rather than relying on representative sections.

For functional studies, combine immunohistochemical detection of KISS1 with assessment of downstream signaling events, such as phosphorylation of ERK1/2 in GnRH neurons or LH release from the pituitary. This antibody can also be valuable in validating kisspeptin neuron-specific transgenic mouse models through confirmation of targeted cell populations. When examining developmental changes in the KISS1 system, note that expression patterns shift significantly during puberty, requiring careful age-matching of experimental groups.

How does the performance of HRP-conjugated KISS1 antibody compare with traditional unconjugated primary antibodies in multiplex immunohistochemistry protocols?

For chromogenic multiplex IHC, the HRP-conjugated antibody should be used for the final antigen in your sequence to avoid potential cross-reactivity with subsequent detection steps. When designing multiplex fluorescent protocols, note that the HRP-conjugation limits your detection options to tyramide-based systems, which may require careful optimization of signal development time to avoid overwhelming other fluorophores in your panel.

What are the critical differences in sample preparation requirements when using KISS1 Antibody, HRP conjugated for different applications (Western blot vs. IHC vs. ELISA)?

Sample preparation requirements vary significantly across applications when using KISS1 Antibody, HRP conjugated. For Western blotting, protein extraction should include strong denaturing conditions (SDS-based buffers) to fully expose the Kisspeptin-10 epitope recognized by this antibody . Include protease inhibitors specifically effective against proteases that process small peptides, and consider phosphatase inhibitors if examining phosphorylation-dependent processing of KISS1.

For immunohistochemistry applications, fixation conditions critically impact epitope preservation. While the antibody works with both frozen and paraffin-embedded tissues , overfixation in formalin can mask the epitope. Limit fixation to 24 hours for optimal results, and implement heat-induced epitope retrieval using citrate buffer (pH 6.0) for paraffin sections. For frozen sections, brief post-fixation in 4% paraformaldehyde (10 minutes) helps preserve tissue morphology while maintaining epitope accessibility.

For ELISA applications, the sample matrix effects vary by sample type. Serum and plasma samples require dilution in buffers containing detergents to reduce matrix interference, while cell culture supernatants may require concentration to detect secreted kisspeptins . When designing ELISA protocols, remember that this antibody recognizes the Kisspeptin-10 region, so assay sensitivity may vary for different processed forms of KISS1. Cross-validate findings from different applications to ensure biological relevance of your observations.

How can KISS1 Antibody, HRP conjugated be utilized in studies investigating the role of kisspeptin in placentation and pregnancy complications?

KISS1 Antibody, HRP conjugated offers valuable applications for investigating kisspeptin's role in placentation and pregnancy complications. Kisspeptin-10, specifically detected by this antibody , has been identified as a paracrine/endocrine regulator that fine-tunes trophoblast invasion during early pregnancy . To study this process, implement a systematic immunohistochemical analysis of placental tissues across gestational ages, focusing on the maternal-fetal interface where invasion regulation is critical.

For studies of pregnancy complications such as preeclampsia or intrauterine growth restriction, the antibody can be used to compare KISS1 expression patterns between normal and pathological placentas. Combined with markers of trophoblast differentiation and invasion (HLA-G, cytokeratins, integrins), this approach can reveal whether dysregulated KISS1 expression correlates with abnormal placentation. The HRP conjugation provides consistent staining across multiple specimens, which is particularly valuable for large-scale comparative studies.

In experimental models, ex vivo placental explant cultures treated with varying oxygen tensions (to mimic hypoxic conditions in pregnancy complications) can be analyzed for KISS1 expression changes using Western blotting with this antibody. For mechanistic insights, complement protein detection with functional assays measuring calcium signaling in trophoblasts following kisspeptin stimulation, as kisspeptin-10 has been shown to increase intracellular Ca²⁺ levels in first-trimester trophoblasts .

What approaches can be used to integrate KISS1 antibody-based detection with transcriptomic data to understand the regulation of KISS1 expression in different physiological contexts?

Integrating KISS1 antibody-based protein detection with transcriptomic data requires strategic experimental design to bridge protein-level and transcript-level insights. Begin with parallel sampling for both analyses: collect adjacent tissue sections or aliquots of the same cell populations for protein detection and RNA extraction. For hypothalamic studies, laser capture microdissection of KISS1-positive neurons (identified using immunohistochemistry on guide sections) followed by RNA-seq provides cell-type-specific transcriptomic data that can be directly correlated with protein expression patterns .

For broader physiological contexts, implement time-course studies across relevant transitions (pubertal development, estrous cycle phases, pregnancy progression) with matched samples for Western blotting using the HRP-conjugated antibody and RNA-seq or qPCR analysis of KISS1 and related transcripts. Discrepancies between mRNA and protein levels may reveal post-transcriptional regulation, which can be further investigated by examining expression of microRNAs predicted to target KISS1.

Advanced integrative approaches include:

• Correlating KISS1 protein levels (quantified by digital image analysis of immunohistochemistry) with transcription factor binding at the KISS1 promoter (from ChIP-seq data)

• Comparing epigenetic modifications at the KISS1 locus (DNA methylation, histone modifications) with protein expression across tissues or disease states

• Using single-cell approaches where possible - combining index sorting of cells followed by scRNA-seq with immunofluorescence quantification of KISS1 protein

These integrative approaches can reveal the multifaceted regulation of KISS1 expression and identify context-specific regulatory mechanisms governing this physiologically important signaling system.