ERLIN2 Antibody, Biotin conjugated

What is ERLIN2 and what are its primary cellular functions?

ERLIN2 (Endoplasmic Reticulum Lipid Raft-Associated Protein 2) is a component of the ERLIN1/ERLIN2 complex that mediates endoplasmic reticulum-associated degradation (ERAD) of inositol 1,4,5-trisphosphate receptors (IP3Rs). The protein plays a critical role in promoting sterol-accelerated ERAD of HMGCR, likely through an AMFR/gp78-containing ubiquitin ligase complex . ERLIN2 is also significantly involved in regulating cellular cholesterol homeostasis through modulation of the SREBP signaling pathway and may promote ER retention of the SCAP-SREBF complex . In cancer biology, ERLIN2 has been identified as a novel oncogenic factor associated with the ER stress response pathway, facilitating adaptation of breast epithelial cells to ER stress by supporting cell growth and protecting cells from ER stress-induced cell death . These diverse functions make ERLIN2 an important target for research in cellular homeostasis and cancer biology.

What specific epitopes do biotin-conjugated ERLIN2 antibodies typically recognize?

Biotin-conjugated ERLIN2 antibodies are designed to recognize specific amino acid sequences within the ERLIN2 protein. Based on current commercially available antibodies, these epitopes commonly include the amino acid region 25-206, which represents a significant portion of the N-terminal domain of the protein . This region is particularly useful for experimental applications as it contains distinctive sequences that can be recognized with high specificity. Other ERLIN2 antibodies (including those with different conjugations) may target alternative regions such as amino acids 47-339, 212-339, or the C-terminal region (amino acids 307-333) . The choice of epitope affects experimental outcomes, as different functional domains of ERLIN2 are involved in various protein-protein interactions and cellular processes.

Why is biotin conjugation useful for ERLIN2 antibody applications?

Biotin conjugation of ERLIN2 antibodies provides significant advantages for various experimental applications due to the strong and specific interaction between biotin and streptavidin/avidin. This conjugation strategy enables amplification of detection signals, enhancing sensitivity in techniques where protein abundance may be limited . The biotin-streptavidin system allows for versatile experimental designs, including multi-step detection protocols, pull-down assays, and immunoprecipitation studies. In immunohistochemistry and immunofluorescence applications, biotin-conjugated antibodies can be used with streptavidin-linked detection systems (enzymes, fluorophores, or gold particles), providing researchers flexibility in visualization methods . Additionally, the biotin tag rarely interferes with antibody binding to the ERLIN2 epitope, maintaining high specificity while adding detection advantages.

How does ERLIN2 relate to breast cancer research?

ERLIN2 has emerged as a significant factor in breast cancer pathobiology. Research has demonstrated that ERLIN2 gene amplification and resulting protein overexpression occur in both luminal and Her2 subtypes of breast cancer . Quantitative genomic PCR and array comparative genomic hybridization (CGH) analyses have shown that the ERLIN2 gene is commonly amplified in approximately 30% of tested breast cancer cell lines and 7.8% of primary breast cancer specimens . The SUM-44, SUM-52 (luminal subtype), and SUM-225 (Her2-amplified) cell lines all display ERLIN2 amplification, demonstrating its relevance across different breast cancer molecular classifications . Functionally, ERLIN2 appears to confer a selective growth advantage by facilitating a cytoprotective response to cellular stresses associated with oncogenesis. The protein supports breast cancer cell adaptation to ER stress by promoting cell growth and protecting against ER stress-induced cell death . These findings position ERLIN2 as a potentially important biomarker and therapeutic target in breast cancer research.

How does the IRE1α/XBP1 signaling axis regulate ERLIN2 expression in breast cancer?

The IRE1α/XBP1 signaling axis exerts significant regulatory control over ERLIN2 protein expression in breast cancer cells, representing a complex interaction between cellular stress response pathways and oncogenic factors. Research has demonstrated that inhibition of IRE1α RNase activity substantially reduces ERLIN2 protein levels in SUM-44 breast cancer cells, establishing a direct regulatory relationship . Conversely, experimental overexpression of wild-type IRE1α or spliced XBP1 in MCF10A mammary epithelial cells results in increased expression of endogenous ERLIN2 protein, further confirming this regulatory connection . Interestingly, quantitative real-time RT-PCR analysis reveals that overexpression of IRE1α or spliced XBP1 does not increase ERLIN2 mRNA levels, suggesting that the regulation occurs at a post-transcriptional level rather than through direct transcriptional activation . This sophisticated regulatory mechanism suggests that ERLIN2 functions as a downstream effector of the IRE1α/XBP1 pathway in the cellular response to endoplasmic reticulum stress, particularly in the context of breast cancer pathophysiology.

How can researchers distinguish between ERLIN1 and ERLIN2 in experimental systems?

Distinguishing between ERLIN1 and ERLIN2 in experimental systems requires careful consideration of antibody selection and validation strategies. While ERLIN1 and ERLIN2 share structural similarities and form functional complexes, they possess distinct amino acid sequences that can be targeted for specific detection . When selecting antibodies, researchers should prioritize those targeting unique epitopes, such as the C-terminal region (amino acids 307-333 for ERLIN2), which typically exhibits greater sequence divergence between the two proteins . Rigorous validation protocols should include Western blot analysis using recombinant ERLIN1 and ERLIN2 proteins to confirm specificity and absence of cross-reactivity. For cell-based assays, siRNA or CRISPR-based knockdown/knockout controls for each protein individually provide critical validation of antibody specificity . When studying protein complexes containing both ERLIN1 and ERLIN2, sequential immunoprecipitation approaches can help delineate the distinct roles of each protein. Additionally, mass spectrometry-based proteomics following immunoprecipitation can confirm antibody specificity by identifying unique peptide sequences for each protein. These comprehensive validation steps ensure reliable discrimination between these related but functionally distinct proteins in experimental systems.

What mechanisms explain ERLIN2's role in protecting cancer cells from ER stress-induced apoptosis?

ERLIN2 employs several sophisticated mechanisms to protect cancer cells from ER stress-induced apoptosis, representing a critical adaptation in tumor biology. As a component of the ERLIN1/ERLIN2 complex, it mediates endoplasmic reticulum-associated degradation (ERAD) of inositol 1,4,5-trisphosphate receptors (IP3Rs), which regulates calcium homeostasis within the ER . Disruption of calcium homeostasis is a known trigger for ER stress-induced apoptosis; therefore, ERLIN2's role in IP3R degradation may prevent catastrophic calcium release during cellular stress. Additionally, ERLIN2 appears to facilitate the activation of the Unfolded Protein Response (UPR) and ERAD pathways, inducing adaptive responses that help tumor cells overcome ER stress through cytoprotective mechanisms . Research indicates that ERLIN2 amplification and overexpression occur in both luminal and HER2 subtypes of breast cancer, suggesting its protective function may be particularly relevant in these cancer contexts . The connection between ERLIN2 and the IRE1α/XBP1 axis of the UPR pathway further supports its role in orchestrating cellular adaptation to stress conditions that would otherwise trigger apoptotic pathways . These multiple protective mechanisms make ERLIN2 a potentially important therapeutic target for strategies aimed at sensitizing cancer cells to ER stress-induced cell death.

How does ERLIN2 contribute to cellular cholesterol homeostasis regulation?

ERLIN2 plays a multifaceted role in regulating cellular cholesterol homeostasis through several interconnected mechanisms. It significantly impacts the SREBP signaling pathway, which is central to cholesterol synthesis regulation, potentially by promoting endoplasmic reticulum retention of the SCAP-SREBF complex . This regulatory action would prevent the nuclear translocation of SREBPs and subsequent activation of genes involved in cholesterol synthesis, thereby functioning as a negative regulator of cholesterol production. Additionally, ERLIN2 promotes sterol-accelerated endoplasmic reticulum-associated degradation (ERAD) of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol biosynthesis . This degradation process likely involves an AMFR/gp78-containing ubiquitin ligase complex, suggesting ERLIN2 participates in coordinating the recognition and ubiquitination of HMGCR in response to elevated sterol levels . The ERLIN1/ERLIN2 complex's localization to lipid rafts in the ER membrane further positions it as a strategic sensor of membrane cholesterol content, potentially allowing it to respond to changes in cholesterol levels and initiate appropriate homeostatic responses . These diverse mechanisms collectively establish ERLIN2 as a critical component in maintaining cellular cholesterol balance, with important implications for both normal physiology and disease states where cholesterol metabolism is dysregulated.

What are the optimal conditions for using biotin-conjugated ERLIN2 antibodies in ELISA applications?

For optimal ELISA performance with biotin-conjugated ERLIN2 antibodies, several critical parameters must be carefully controlled. The recommended working dilution should be determined experimentally for each specific application, typically starting with a range of 1:500 to 1:5000 for initial optimization . Sample preparation is crucial - for cell lysates, using RIPA buffer supplemented with protease inhibitors ensures protein integrity while providing effective extraction of membrane-associated ERLIN2. When coating ELISA plates, a carbonate/bicarbonate buffer (pH 9.6) is recommended for optimal protein binding, with overnight incubation at 4°C to maximize coating efficiency . Blocking solutions containing 2-5% BSA or casein in PBS-T (PBS with 0.05% Tween-20) effectively minimize non-specific binding without interfering with the biotin-streptavidin interaction. For detection, high-quality streptavidin-HRP conjugates used at 1:10,000-1:20,000 dilution provide optimal signal-to-noise ratios. Washing steps should be rigorous (4-5 washes with PBS-T) to reduce background while preserving specific signal. Temperature control is also important - all incubation steps should be performed at room temperature (22-25°C) except for the initial plate coating. Finally, validation with appropriate positive controls (recombinant ERLIN2 protein) and negative controls (non-specific IgG) is essential for result interpretation .

What sample preparation techniques yield optimal results for detecting ERLIN2 in breast cancer tissues?

Effective detection of ERLIN2 in breast cancer tissues requires specialized sample preparation techniques that preserve both protein integrity and tissue architecture. For formalin-fixed paraffin-embedded (FFPE) samples, optimal antigen retrieval involves heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 20 minutes, with the specific buffer choice depending on the particular epitope targeted by the antibody . Fresh tissue samples should be fixed in 10% neutral-buffered formalin for 24-48 hours to maintain structural integrity while enabling antibody penetration. For protein extraction from tissues intended for immunoblotting, specialized extraction buffers containing both ionic and non-ionic detergents (such as modified RIPA buffer with 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) are recommended to solubilize membrane-associated ERLIN2 . Protein extraction should be performed at 4°C with mechanical homogenization followed by sonication to maximize yield. For immunohistochemical applications, 3-5 μm tissue sections provide optimal thickness, and biotin-blocking steps are essential when using biotin-conjugated antibodies to prevent non-specific binding, particularly in tissues with high endogenous biotin content such as breast tissues . Finally, for samples with both ERLIN2 and Her2 amplification, sequential staining protocols with careful intermediate stripping or blocking steps help prevent cross-reaction when performing multiplex detection .

How can researchers quantitatively measure ERLIN2 expression levels in cell lines with gene amplification?

Quantitative measurement of ERLIN2 expression in cell lines with gene amplification requires a multi-faceted approach combining genomic, transcriptomic, and proteomic techniques. For genomic quantification, quantitative PCR (qPCR) with primers targeting the ERLIN2 gene region allows determination of copy number variations relative to reference genes . Digital droplet PCR provides even higher precision for samples with heterogeneous amplification patterns. At the transcriptional level, quantitative real-time RT-PCR using validated ERLIN2-specific primers enables measurement of mRNA expression, though research indicates that mRNA levels may not always correlate directly with protein abundance, particularly in the context of IRE1α/XBP1 regulation . For protein quantification, quantitative Western blotting with infrared fluorescence-based detection systems provides superior linearity compared to chemiluminescence methods. Cell lysates should be prepared using standardized protocols, and protein loading must be carefully normalized using multiple housekeeping proteins such as GAPDH and β-actin . For higher throughput analysis, flow cytometry with permeabilized cells and fluorophore-conjugated ERLIN2 antibodies enables rapid quantification across numerous samples. Finally, absolute quantification can be achieved using recombinant ERLIN2 protein standards to generate calibration curves for Western blot or ELISA-based assays . This comprehensive approach provides researchers with robust quantitative data on ERLIN2 expression across different experimental systems and cancer cell lines.

What controls are essential when using biotin-conjugated ERLIN2 antibodies in immunohistochemistry?

Implementing rigorous controls is essential for reliable immunohistochemistry (IHC) results when using biotin-conjugated ERLIN2 antibodies. Positive tissue controls should include samples with confirmed ERLIN2 expression, such as SUM-44, SUM-52, or SUM-225 breast cancer cell lines, which demonstrate high ERLIN2 protein levels correlating with gene amplification . Negative tissue controls should include samples known to have minimal ERLIN2 expression or tissues from ERLIN2 knockout models when available. Procedural controls are equally critical: endogenous biotin blocking is mandatory using commercial biotin-blocking kits to prevent false-positive signals, particularly in biotin-rich tissues such as liver, kidney, and some breast cancers . Isotype controls (biotin-conjugated non-specific IgG of the same host species and isotype) help distinguish specific staining from background caused by non-specific antibody binding . Absorption controls, where the antibody is pre-incubated with recombinant ERLIN2 protein before application to tissue, confirm binding specificity. Signal development controls without primary antibody but with streptavidin-detection systems verify the absence of non-specific binding of the detection reagents. Finally, when evaluating tissues with potential ERLIN2 and Her2 co-amplification, parallel staining with Her2-specific antibodies on sequential sections provides important context for interpretation . These comprehensive controls ensure reliable and reproducible IHC results when investigating ERLIN2 expression in research and potential diagnostic applications.

What is the optimal approach for investigating ERLIN2's interaction with the IRE1α/XBP1 pathway?

Investigating ERLIN2's interaction with the IRE1α/XBP1 pathway requires a sophisticated experimental approach combining molecular, cellular, and biochemical techniques. Co-immunoprecipitation experiments using biotin-conjugated ERLIN2 antibodies followed by streptavidin pull-down can capture protein complexes containing ERLIN2, IRE1α, and other pathway components for subsequent analysis by Western blotting or mass spectrometry . Proximity ligation assays (PLA) provide spatial resolution of protein interactions within intact cells, detecting ERLIN2-IRE1α interactions at their native subcellular locations. For functional studies, siRNA or CRISPR-Cas9 mediated knockdown/knockout of ERLIN2 followed by analysis of IRE1α phosphorylation status and XBP1 splicing helps establish pathway dependencies . Conversely, overexpression of wild-type IRE1α or spliced XBP1 with subsequent measurement of ERLIN2 protein levels (but not mRNA, based on previous findings) elucidates regulatory relationships . Reporter assays using XBP1 splicing-dependent luciferase constructs can quantitatively measure pathway activation in response to ERLIN2 modulation. For dynamic studies, time-course experiments following ER stress induction (using tunicamycin or thapsigargin) in cells with normal or altered ERLIN2 expression reveal temporal relationships between pathway components. Finally, domain mapping experiments using truncated ERLIN2 constructs help identify specific protein regions required for interaction with the IRE1α/XBP1 pathway . This comprehensive approach provides mechanistic insights into ERLIN2's role in this critical stress response pathway.

How can researchers address weak signal problems when using biotin-conjugated ERLIN2 antibodies?

When encountering weak signal issues with biotin-conjugated ERLIN2 antibodies, researchers should implement a systematic troubleshooting approach. First, titration experiments with decreasing antibody dilutions (starting from 1:500 and gradually increasing concentration) can identify optimal working concentrations for specific experimental conditions . Enhanced antigen retrieval protocols may be necessary - for FFPE samples, extending heat-induced epitope retrieval time to 30 minutes or exploring alternative buffers such as high-pH EDTA buffer (pH 9.0) can significantly improve epitope accessibility . Signal amplification systems such as tyramide signal amplification (TSA) can dramatically enhance sensitivity without increasing background when properly optimized. For immunoblotting applications, membrane transfer efficiency should be verified using reversible stains like Ponceau S before immunodetection, as ERLIN2's membrane-associated nature can sometimes result in inefficient transfer . Sample preparation modifications, including longer extraction times, stronger detergent formulations (e.g., adding 0.5% sodium deoxycholate to extraction buffers), or brief sonication can improve ERLIN2 solubilization from membrane fractions . For biotin-based detection systems, extending the streptavidin-enzyme conjugate incubation time and optimizing the substrate development period can enhance signal without introducing excessive background. Finally, antibody storage conditions should be verified, as improper handling or repeated freeze-thaw cycles can diminish biotin conjugation integrity and reduce effective antibody concentration .

What strategies can resolve high background issues in ERLIN2 immunodetection experiments?

Resolving high background issues in ERLIN2 immunodetection requires targeted interventions addressing multiple potential causes. For immunohistochemistry and immunofluorescence applications, implementing comprehensive biotin blocking is crucial, particularly in tissues with high endogenous biotin (like breast tissue), using commercial avidin/biotin blocking kits before applying biotin-conjugated antibodies . Optimizing blocking solutions by using 5% BSA or 5% normal serum from the same species as the secondary reagent significantly reduces non-specific binding. For Western blotting, increasing blocking stringency with 5% non-fat dry milk in TBS-T (0.1% Tween-20) followed by extensive washing steps (5 × 5 minutes with TBS-T) effectively reduces background . Reducing primary antibody concentration through careful titration experiments often improves signal-to-noise ratio, as excess antibody contributes significantly to background issues . For all applications, using freshly prepared reagents and filtered buffers eliminates contamination-related background. Secondary detection reagent optimization is equally important - using highly purified streptavidin conjugates at dilutions ranging from 1:2000 to 1:5000 and shortening incubation times can substantially reduce non-specific binding. When persistent background issues occur, substituting alternative detection systems (such as directly conjugated primary antibodies or non-biotin amplification systems) may be necessary. Finally, including procedural controls without primary antibody but with complete detection systems helps distinguish between background caused by primary versus secondary detection components .

How should researchers validate the specificity of ERLIN2 antibody detection in experimental systems?

Comprehensive validation of ERLIN2 antibody specificity requires a multi-faceted approach employing several complementary strategies. Genetic validation using CRISPR-Cas9 knockout or siRNA knockdown of ERLIN2 followed by immunodetection provides the most definitive specificity confirmation, as the specific signal should be substantially reduced or eliminated in these samples . Peptide competition assays, where the antibody is pre-incubated with increasing concentrations of the immunizing peptide or recombinant ERLIN2 protein before application to samples, should show dose-dependent signal reduction if the antibody is specific . Multiple-antibody validation using antibodies targeting different ERLIN2 epitopes (such as N-terminal versus C-terminal regions) should yield concordant detection patterns across different techniques . Western blot analysis should demonstrate a predominant band at the expected molecular weight of ERLIN2 (~38 kDa), with minimal non-specific bands. For biotin-conjugated antibodies specifically, parallel experiments with unconjugated versions of the same antibody clone can help distinguish biotin-related artifacts from true ERLIN2 detection . Cross-reactivity assessment with related proteins, particularly ERLIN1, is essential given their structural similarity; this can be accomplished through parallel detection of recombinant ERLIN1 and ERLIN2 proteins . Finally, correlation of protein detection with genomic amplification status in characterized cell lines (such as SUM-44, SUM-52, and SUM-225) provides additional validation of specificity in biological contexts where ERLIN2 is known to be overexpressed .

What factors affect the reproducibility of ERLIN2 detection across different experimental batches?

Multiple factors influence the reproducibility of ERLIN2 detection across experimental batches, requiring careful standardization for consistent results. Antibody lot-to-lot variation represents a significant challenge; researchers should record lot numbers and perform validation experiments when switching to new antibody batches . Storage conditions significantly impact biotin-conjugated antibodies - they should be stored at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can degrade both the antibody and the biotin conjugation . Sample preparation standardization is critical; consistent cell lysis protocols, protein extraction methods, and protein quantification techniques ensure comparable starting material across experiments . For immunohistochemistry applications, fixation time, processing protocols, and antigen retrieval conditions must be rigorously controlled, as variations directly impact epitope availability . Instrument calibration for detection systems (plate readers, flow cytometers, imaging systems) should be performed regularly using standardized calibration tools. Environmental factors including temperature and humidity affect reaction kinetics and should be controlled, particularly for enzymatic detection systems. Reference standards, including recombinant ERLIN2 protein at known concentrations, should be included in each experimental batch to enable normalization across experiments . Finally, implementing detailed standard operating procedures (SOPs) for all experimental steps and using automated systems where possible (automated washers, liquid handlers, staining platforms) significantly enhances reproducibility by minimizing operator-dependent variations .

How can researchers distinguish between true ERLIN2 signal and artifacts in samples with endogenous biotin?

Distinguishing true ERLIN2 signal from endogenous biotin artifacts requires implementation of specialized technical approaches and critical controls. Prior to applying biotin-conjugated antibodies, comprehensive biotin blocking is essential, using commercial avidin/biotin blocking kits with sequential application of avidin (to bind endogenous biotin) followed by biotin (to saturate remaining avidin binding sites) . Parallel staining with non-biotinylated ERLIN2 antibodies targeting the same epitope provides a critical comparison to identify potential biotin-related artifacts. For tissues known to have high endogenous biotin levels (liver, kidney, and some breast cancer samples), alternative detection systems such as directly conjugated primary antibodies or polymer-based detection methods should be considered . Pre-treatment of tissue sections with dilute hydrogen peroxide (0.3% H₂O₂ in methanol) for 30 minutes prior to standard blocking steps can help quench endogenous peroxidase activity that might otherwise contribute to false signals when using HRP-based detection systems. Control slides processed without primary antibody but with complete streptavidin-based detection systems help identify non-specific streptavidin binding sites. Serial dilution experiments with primary antibodies should show proportional signal reduction with true ERLIN2 detection, while biotin artifacts typically show threshold effects . Finally, correlation with orthogonal detection methods (such as in situ hybridization for ERLIN2 mRNA or mass spectrometry) provides independent confirmation of ERLIN2 expression patterns without reliance on biotin-based systems .