PELP1 Recombinant Monoclonal Antibody

What is PELP1 and what are its primary cellular functions?

PELP1, also known as Modulator of Nongenomic Activity of Estrogen Receptor (MNAR), is a 120 kDa scaffolding protein that plays significant roles in chromatin remodeling and transcriptional regulation. PELP1 functions as a coactivator of estrogen receptor-mediated transcription while serving as a corepressor for other nuclear hormone receptors and sequence-specific transcription factors . In the nuclear compartment, PELP1 activates estrogen receptor target genes in a hormone-dependent manner, while in the cytosol, it facilitates estrogen receptor non-genomic signaling via SRC and PI3K interactions . This protein also participates in E2-mediated cell cycle progression through interactions with RB1 and plays a critical role in ER/growth factor cross-talk . Recent studies have revealed PELP1's involvement in angiogenesis regulation in colorectal cancer via the STAT3/VEGFA axis, suggesting its potential as a therapeutic target .

What applications are PELP1 recombinant monoclonal antibodies suitable for?

PELP1 recombinant monoclonal antibodies demonstrate versatility across multiple laboratory applications. These antibodies are validated for Western blotting (WB), with recommended dilutions typically ranging from 1:500-1:5000, allowing researchers to detect endogenous PELP1 protein in cell and tissue lysates . They are also suitable for immunocytochemistry and immunofluorescence (ICC/IF) applications at dilutions of approximately 1:50-1:200, enabling visualization of PELP1's subcellular localization . Flow cytometry (intracellular) applications are supported by certain antibody clones, providing a means to quantify PELP1 expression at the single-cell level . Additionally, some PELP1 antibodies are validated for enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP), expanding their utility for protein interaction studies . When selecting an antibody, researchers should consider the specific clone and validation data for their intended application.

What is the optimal storage and handling protocol for PELP1 antibodies?

Proper storage and handling of PELP1 recombinant monoclonal antibodies are essential for maintaining their activity and specificity. Most manufacturers recommend storing these antibodies at 4°C for short-term use and at -20°C or -80°C for long-term storage . To preserve antibody integrity, it's advisable to aliquot the stock solution to avoid repeated freeze-thaw cycles, which can degrade protein structure and reduce antibody efficacy . The typical storage buffer consists of 50mM Tris-Glycine, 150mM NaCl, pH 7.4, with 40% glycerol, 0.05% BSA, and 0.01% sodium azide as preservatives . Researchers should note that sodium azide is toxic and should be handled with appropriate safety precautions . For optimal performance, antibodies should be thawed completely before use and gently mixed to ensure homogeneity. Following the manufacturer's recommended working dilutions for specific applications is crucial for achieving reliable and reproducible results.

What species reactivity is exhibited by PELP1 recombinant monoclonal antibodies?

The species reactivity of PELP1 recombinant monoclonal antibodies varies depending on the specific clone and manufacturer. Based on the available data, most PELP1 antibodies demonstrate strong reactivity with human PELP1 . Some antibody clones, particularly the E-1 mouse monoclonal antibody from Santa Cruz Biotechnology, show cross-reactivity with mouse and rat PELP1 in addition to human PELP1 . This cross-species reactivity makes these antibodies valuable for comparative studies across different model organisms. The species specificity stems from the immunogen used during antibody production, which is typically a synthetic peptide derived from human PELP1 . Researchers should carefully review the manufacturer's validation data for species reactivity when selecting an antibody for experiments involving non-human samples, as performance may vary significantly between species despite sequence homology.

How can PELP1 antibodies be used to investigate estrogen receptor signaling pathways?

PELP1 antibodies serve as powerful tools for dissecting the complex interplay between PELP1 and estrogen receptor (ER) signaling pathways. To study genomic ER signaling, researchers can employ chromatin immunoprecipitation (ChIP) assays using PELP1 antibodies to identify genomic regions where PELP1 and ERs co-localize, revealing target genes under hormonal control . For non-genomic ER signaling investigation, co-immunoprecipitation experiments with PELP1 antibodies can capture protein complexes containing PELP1, SRC, and PI3K, illuminating cytoplasmic signaling cascades . Immunofluorescence microscopy using PELP1 antibodies enables visualization of PELP1 translocation between nuclear and cytoplasmic compartments in response to estrogen stimulation, providing insights into spatiotemporal dynamics . When designing such experiments, researchers should include appropriate controls, such as hormone-depleted conditions (using charcoal-stripped serum) and time-course analyses following estrogen treatment. The combination of PELP1 antibodies with phospho-specific antibodies targeting key signaling molecules can further elucidate PELP1's role in activating downstream pathways like MAPK and Akt, which are critical for estrogen's proliferative effects.

What is the role of PELP1 in cancer angiogenesis and how can it be studied using PELP1 antibodies?

PELP1 has emerged as a key regulator of angiogenesis in cancer, particularly in colorectal cancer (CRC), where its expression positively correlates with microvessel density (MVD) . To investigate this function, researchers can utilize PELP1 antibodies in multiple experimental approaches. Immunohistochemistry with PELP1 antibodies on clinical CRC samples, along with endothelial markers like CD31, can establish correlations between PELP1 expression and tumor vascularization . In vitro studies involving PELP1 knockdown or overexpression, followed by Western blot analysis using PELP1 antibodies, can confirm manipulation of protein levels before assessing angiogenic potential through endothelial cell tube formation assays . To elucidate mechanism, PELP1 antibodies can be employed in immunoblotting to monitor changes in STAT3 phosphorylation and VEGFA production following PELP1 modulation, as PELP1 regulates angiogenesis via the STAT3/VEGFA axis . For in vivo validation, xenograft models with PELP1-manipulated cells can be analyzed using immunohistochemistry with PELP1 antibodies to correlate PELP1 expression with tumor growth and vascular parameters. Recent research demonstrated that PELP1 depletion inhibited cell proliferation in HCT116 and HT29, two colorectal cancer cell lines with moderate PELP1 expression, establishing a foundation for future angiogenesis studies .

What technical considerations should be addressed when using PELP1 antibodies for protein-protein interaction studies?

When employing PELP1 antibodies for protein-protein interaction studies, several technical considerations are critical for generating reliable and reproducible results. For co-immunoprecipitation experiments, researchers should optimize lysis conditions to preserve native protein complexes; a buffer containing 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, and protease inhibitors is often suitable for PELP1 interactions . The antibody-to-lysate ratio requires careful titration, with typical starting points being 2-5 μg of antibody per 500 μg of total protein. When performing proximity ligation assays (PLA) to visualize PELP1 interactions in situ, fixation protocols must be optimized to maintain epitope accessibility while preserving cellular architecture; 4% paraformaldehyde for 15 minutes at room temperature is generally effective . For pull-down assays with recombinant PELP1, researchers should be aware that the large size of PELP1 (120 kDa) may impact protein folding and interaction capacity, necessitating quality control steps to verify proper conformation . Controls are crucial in all interaction studies: isotype-matched control antibodies should be used for immunoprecipitation, while competitive blocking with immunizing peptides can confirm antibody specificity . Additionally, bidirectional immunoprecipitation (pulling down with antibodies against both PELP1 and its suspected binding partner) strengthens confidence in identified interactions.

How should researchers validate PELP1 antibody specificity in their experimental systems?

Rigorous validation of PELP1 antibody specificity is essential for ensuring experimental reliability. A comprehensive validation approach involves multiple complementary strategies. First, researchers should perform Western blot analysis across multiple cell lines with known PELP1 expression levels (e.g., HeLa, A549, and HL-60) to verify that the antibody detects a single band at the expected molecular weight of approximately 120 kDa . Genetic validation using PELP1 knockdown (siRNA or shRNA) or knockout (CRISPR-Cas9) systems provides compelling evidence of specificity, as the signal should diminish proportionally to reduction in PELP1 expression . For immunocytochemistry applications, parallel staining with two different PELP1 antibodies recognizing distinct epitopes should yield consistent localization patterns . Peptide competition assays, where the immunizing peptide blocks antibody binding, offer additional confirmation of specificity; some manufacturers provide neutralizing peptides specifically for this purpose . Cross-reactivity testing against closely related proteins or in samples from knockout models (where available) provides the gold standard for specificity assessment. Researchers should also be mindful of potential post-translational modifications of PELP1 that might affect antibody recognition, particularly phosphorylation events that occur during signaling activation . Documentation of these validation efforts is crucial for publication and should include images of full Western blots with molecular weight markers.

What are the considerations for using PELP1 antibodies in multiparametric flow cytometry?

When incorporating PELP1 antibodies into multiparametric flow cytometry panels, researchers must address several technical aspects to achieve robust data. Since PELP1 is predominantly an intracellular protein, effective cell fixation and permeabilization are critical; a protocol using 4% paraformaldehyde followed by 0.1% Triton X-100 or commercially available permeabilization kits designed for nuclear proteins typically yields good results . Titration of the PELP1 antibody is essential to determine the optimal concentration that maximizes signal-to-noise ratio; this should be performed in the context of the full antibody panel to account for potential spectral overlap. For multicolor panels, researchers should select a fluorophore for PELP1 detection that avoids spectral overlap with markers of interest in their cellular system. Compensation controls, including single-stained and fluorescence-minus-one (FMO) controls, are mandatory for accurate data interpretation. When studying PELP1 in relation to cell cycle or proliferation, co-staining with DNA content markers (e.g., DAPI or propidium iodide) and proliferation markers (e.g., Ki-67) can provide valuable contextual information . For analyzing PELP1 in the context of estrogen signaling, researchers might consider including phospho-specific antibodies against downstream signaling molecules like phospho-ERK or phospho-Akt in their panel. Careful selection of blocking agents during sample preparation is important, as some blocking solutions may interfere with epitope recognition by the PELP1 antibody.

What are common issues encountered when using PELP1 antibodies in Western blotting and how can they be resolved?

When using PELP1 antibodies for Western blotting, researchers frequently encounter several technical challenges. Background noise is a common issue that can obscure specific PELP1 detection at 120 kDa. This can be mitigated by increasing blocking stringency (5% BSA or milk in TBST for 1-2 hours at room temperature), optimizing antibody dilution (typically between 1:500-1:5000), and extending washing steps (4-5 washes of 10 minutes each) . Weak or absent PELP1 signal may result from inefficient protein extraction, as PELP1 can associate with chromatin in the nucleus; researchers should consider using RIPA buffer supplemented with nucleases or specialized nuclear extraction kits . Multiple bands or unexpected molecular weight patterns might indicate protein degradation or post-translational modifications; adding protease inhibitor cocktails freshly to lysis buffers and keeping samples cold throughout processing can preserve protein integrity . For difficult-to-detect PELP1, signal enhancement systems or more sensitive detection methods like chemiluminescence substrates with extended signal duration may prove beneficial. Non-specific binding can be reduced by pre-adsorbing the antibody with non-relevant proteins or using highly purified antibody preparations. Testing different PELP1 antibody clones is advisable if persistent issues occur, as epitope accessibility can vary between applications and experimental conditions .

How can researchers optimize immunohistochemistry protocols for PELP1 detection in tissue samples?

Optimizing immunohistochemistry (IHC) protocols for PELP1 detection in tissue samples requires systematic refinement of multiple parameters. Antigen retrieval is particularly critical for PELP1 detection; heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) at 95-98°C for 20-30 minutes typically yields good results . The optimal antibody dilution range for IHC applications is generally between 1:50-1:200, but this should be determined empirically for each tissue type and fixation method . Blocking endogenous peroxidase activity (3% H₂O₂ for 10 minutes) and non-specific binding sites (5-10% normal serum from the same species as the secondary antibody) minimizes background staining. Incubation conditions significantly impact staining quality; overnight incubation at 4°C often produces more specific staining compared to shorter incubations at room temperature . For formalin-fixed paraffin-embedded tissues, section thickness (4-5 μm is optimal) and fixation duration affect antibody penetration and epitope preservation. Positive controls (tissues known to express PELP1, such as breast or endometrial cancer samples) and negative controls (omission of primary antibody or use of isotype control) are essential for validating staining specificity . Signal amplification systems like polymer-based detection kits can enhance sensitivity without increasing background. When analyzing results, researchers should note that PELP1 may exhibit both nuclear and cytoplasmic localization depending on the tissue type and pathological state, with subcellular distribution potentially carrying prognostic significance .

What strategies can improve detection of PELP1 in co-immunoprecipitation experiments?

Enhancing PELP1 detection in co-immunoprecipitation (co-IP) experiments requires strategies that preserve protein interactions while maximizing specificity. The choice of lysis buffer is crucial; a buffer containing 20mM Tris-HCl (pH 7.5), 150mM NaCl, 1% NP-40 or Triton X-100, 1mM EDTA, and protease/phosphatase inhibitors preserves most PELP1 interactions while efficiently solubilizing membrane-associated complexes . Cross-linking proteins in situ with membrane-permeable crosslinkers (such as DSP or formaldehyde at 0.5-1%) before lysis can stabilize transient interactions involving PELP1. Pre-clearing lysates with protein A/G beads reduces non-specific binding to the beads themselves. The antibody-to-lysate ratio should be optimized through titration experiments; generally, 2-5 μg of PELP1 antibody per 500-1000 μg of total protein provides a good starting point . Sequential co-IP approaches, where the first IP isolates PELP1 complexes and a second IP targets suspected interaction partners, can validate specific interactions. For challenging co-IPs, adjusting salt concentration (typically 100-250mM NaCl) can modulate stringency, balancing between preserving interactions and reducing background. When detecting PELP1 in immunoprecipitated complexes by Western blot, using a different PELP1 antibody clone recognizing a distinct epitope from that used for IP avoids detection of heavy and light chains that may obscure the PELP1 signal . Including appropriate controls, such as IgG-matched negative controls and input lysate lanes (typically 5-10% of IP input), is essential for accurate interpretation of results.

How can PELP1 antibodies contribute to research on cancer therapy resistance mechanisms?

PELP1 antibodies offer valuable tools for investigating therapy resistance mechanisms in cancer. In hormone-dependent cancers like breast cancer, immunohistochemistry with PELP1 antibodies can be used to analyze cytoplasmic versus nuclear localization shifts, as altered PELP1 localization correlates with tamoxifen resistance . Western blotting with PELP1 antibodies before and after therapeutic challenges allows monitoring of expression changes associated with acquired resistance, particularly in estrogen receptor-positive tumors undergoing endocrine therapy . Co-immunoprecipitation experiments using PELP1 antibodies can identify novel protein interactions that emerge during resistance development, potentially revealing bypass signaling pathways . For mechanistic studies, chromatin immunoprecipitation (ChIP) with PELP1 antibodies can detect altered genomic binding patterns in resistant versus sensitive cells, indicating reprogrammed transcriptional networks . In colorectal cancer models, PELP1 antibodies have helped establish that suppression of PELP1 enhances chemotherapy efficacy through vascular normalization, suggesting combination approaches targeting PELP1 might overcome treatment resistance . Researchers investigating PI3K/Akt inhibitor resistance should consider examining PELP1's role, as it facilitates crosstalk between estrogen receptor and growth factor signaling pathways . For translational research, tissue microarray analysis with PELP1 antibodies can correlate expression patterns with clinical outcomes and treatment responses across large patient cohorts, potentially identifying predictive biomarkers for therapy selection.

What are emerging applications of PELP1 antibodies in studying non-canonical functions of PELP1?

PELP1 antibodies are increasingly being employed to explore non-canonical functions of this versatile protein beyond its established role in estrogen receptor signaling. Immunofluorescence microscopy with PELP1 antibodies has revealed its localization to the nucleolus, where PELP1 participates in ribosomal RNA processing as part of the PELP1 complex involved in 28S rRNA maturation and pre-60S ribosomal subunit transit . Proximity ligation assays using PELP1 antibodies can visualize interactions with MDN1, a critical remodeling factor in ribosome biogenesis, providing spatial context for these functional associations . In the field of epigenetics, ChIP-seq employing PELP1 antibodies can map global chromatin binding patterns, uncovering PELP1's involvement in the Five Friends of Methylated CHTOP (5FMC) complex that regulates ZNF148 target genes through desumoylation mechanisms . For cytoskeletal studies, co-immunoprecipitation with PELP1 antibodies followed by mass spectrometry has identified interactions with cytoskeletal regulators that influence cell migration and metastasis . Western blotting with PELP1 antibodies in fractionated cell compartments can track PELP1's distribution between membrane, cytosolic, and nuclear fractions under various stimuli, revealing condition-specific relocalization . These diverse applications highlight PELP1's multifunctional nature and suggest that PELP1 antibodies will continue to be essential tools as research expands into novel areas such as RNA metabolism, DNA damage response, and metabolic regulation where PELP1 may play previously unappreciated roles.

How might PELP1 antibodies be utilized in developing targeted cancer therapies?

PELP1 antibodies hold significant potential for advancing targeted cancer therapies through multiple research applications. In preclinical drug development, high-throughput screening assays incorporating PELP1 antibodies can identify compounds that disrupt PELP1's protein-protein interactions or alter its subcellular localization, potentially yielding novel therapeutic candidates . Immunohistochemistry with PELP1 antibodies enables patient stratification in clinical trials, selecting those with high PELP1 expression who might benefit most from therapies targeting PELP1-dependent pathways . For developing antibody-drug conjugates, PELP1 antibodies with high specificity and cell-penetrating properties could serve as delivery vehicles for cytotoxic payloads to PELP1-overexpressing cancer cells. In vivo imaging studies using fluorescently labeled or radiolabeled PELP1 antibodies can help assess tumor targeting efficiency and biodistribution of potential therapeutics . Mechanistic studies have shown that targeting PELP1 attenuates angiogenesis in colorectal cancer models, suggesting PELP1 antibodies could help validate anti-angiogenic therapeutic strategies . For combination therapy approaches, PELP1 antibodies can monitor how PELP1 expression or localization changes in response to standard treatments, identifying optimal timing for sequential therapy . As PELP1 promotes tumorigenesis through interactions with multiple oncogenic pathways (SRC, PI3K, STAT3, EGFR), antibodies recognizing specific phosphorylated forms of PELP1 could help monitor pathway activation in response to targeted therapies . Additionally, research using PELP1 antibodies has demonstrated that PELP1 inhibition enhances chemotherapy efficacy through vascular normalization, providing rationale for developing PELP1-targeting agents as chemosensitizers .