ERGIC2 Antibody, HRP conjugated

What is ERGIC2 protein and why is it an important research target?

ERGIC2 (Endoplasmic Reticulum-Golgi Intermediate Compartment protein 2) plays a critical role in transport between the endoplasmic reticulum and Golgi apparatus. This protein, also known as PTX1, Erv41, or CDA14, is involved in maintaining the structural and functional integrity of the early secretory pathway . The protein's function in vesicular trafficking makes it relevant to studies of protein secretion, membrane biogenesis, and organelle homeostasis. Understanding ERGIC2 function has implications for research on neurodegenerative diseases, cancer progression, and cellular stress responses, as disruptions in ER-Golgi trafficking are implicated in these pathological conditions. Most ERGIC2 antibodies target specific amino acid sequences, with commercially available options targeting regions such as AA 252-301, which shows high conservation across multiple species .

How does HRP conjugation enhance detection capabilities in ERGIC2 antibody applications?

Horseradish peroxidase (HRP) conjugation provides a direct enzymatic detection system that catalyzes the conversion of various substrates into colored, fluorescent, or chemiluminescent products. In ERGIC2 antibody applications, this conjugation eliminates the need for secondary antibody incubation steps, thereby reducing background noise, decreasing total assay time, and potentially increasing sensitivity . The HRP enzyme maintains activity in various buffer conditions (pH 6.5-8.5) and provides a versatile detection platform compatible with chromogenic substrates (TMB, DAB), chemiluminescent substrates (luminol-based), and fluorescent tyramide amplification systems . The directional covalent bonding of HRP to the antibody, as achieved in proper conjugation protocols, ensures consistent enzyme-to-antibody ratios, allowing for more quantitative and reproducible results in assays targeting ERGIC2 .

What are the recommended storage and handling conditions for ERGIC2 Antibody, HRP conjugated?

ERGIC2 Antibody, HRP conjugated should be stored at either -20°C or -80°C for long-term preservation of activity . When handling the antibody, several critical considerations should be observed: (1) Avoid repeated freeze-thaw cycles which can diminish activity; (2) Prepare working aliquots upon first thaw; (3) Protect from prolonged exposure to light as HRP is light-sensitive; (4) Sodium azide, a common antibody preservative, is an irreversible inhibitor of HRP and must be strictly avoided in all buffers and solutions ; (5) For working solutions, use 10-50mM amine-free buffers (HEPES, MES, MOPS or phosphate) with pH range 6.5-8.5; (6) Limit exposure to nucleophilic components such as primary amines and thiols which may interfere with the conjugate chemistry ; and (7) Always wear gloves when handling to prevent contamination with peroxidases present on human skin.

How can optimal signal-to-noise ratios be achieved when using ERGIC2 Antibody, HRP conjugated in ELISA?

Achieving optimal signal-to-noise ratios with ERGIC2 Antibody, HRP conjugated requires systematic optimization across multiple parameters. Begin by conducting an antibody titration matrix (typically testing concentrations from 0.1-10 μg/mL) against known positive and negative control samples to identify the optimal concentration that maximizes signal while minimizing background . Buffer optimization is equally critical—using 10-50mM amine-free buffers (HEPES, MES, MOPS, phosphate) at pH 6.5-8.5 can dramatically improve specific binding . Blocking solutions should be carefully selected; 1-5% BSA or casein in PBS/TBS typically outperforms serum-based blockers for HRP-conjugated antibodies.

For washing steps, incorporate 0.05-0.1% Tween-20 in wash buffers and increase wash cycles (5-7 times) between steps to reduce non-specific binding. Substrate selection and development timing significantly impact signal-to-noise ratio—TMB substrates provide better discrimination for quantitative ELISAs, while chemiluminescent substrates offer superior sensitivity for detecting low-abundance ERGIC2. Finally, implement rigorous plate layout design that includes concentration-matched isotype controls, no-primary controls, and no-sample controls to accurately differentiate true signal from background noise .

What validation approaches should be employed to verify ERGIC2 Antibody, HRP conjugated specificity?

Comprehensive validation of ERGIC2 Antibody, HRP conjugated requires implementing multiple orthogonal approaches. First, conduct peptide competition assays using the immunizing peptide (AA 252-301 for some products) to confirm binding specificity—pre-incubation with the peptide should abolish signal . Knockout/knockdown validation provides the gold standard—compare signal between ERGIC2 knockout/knockdown cells and wild-type controls, where specific antibodies should show dramatically reduced signal in ERGIC2-deficient samples. Cross-species reactivity testing is valuable given the high conservation of ERGIC2 across species (100% identity across many mammals); consistent detection patterns across predicted reactive species supports specificity .

For functional validation, analyze subcellular localization patterns—ERGIC2 should demonstrate characteristic perinuclear and punctate cytoplasmic staining consistent with ER-Golgi intermediate compartment distribution. Additionally, perform parallel detection with alternative antibodies targeting different ERGIC2 epitopes (N-terminal, middle region, C-terminal) and compare localization patterns . Finally, mass spectrometry identification of immunoprecipitated proteins provides definitive validation—immunoprecipitate lysates with the ERGIC2 antibody and confirm captured proteins by mass spectrometry to verify that ERGIC2 and known interaction partners are specifically enriched.

What technical considerations are important when switching from unconjugated to HRP-conjugated ERGIC2 antibodies?

Transitioning from unconjugated to HRP-conjugated ERGIC2 antibodies necessitates several methodological adjustments. First, recalibrate antibody concentrations—HRP-conjugated antibodies typically require 2-5 fold lower concentrations than unconjugated versions due to direct detection capability . Incubation protocols must be modified; HRP-conjugated antibodies often require shorter incubation times (1-2 hours at room temperature rather than overnight at 4°C) to minimize nonspecific binding while maintaining detection sensitivity. Buffer systems require particular attention—eliminate any sodium azide, which irreversibly inhibits HRP activity, and avoid primary amines and thiols that can interfere with the conjugate chemistry .

The elimination of secondary antibody steps requires adjustment of washing protocols; increase wash cycles (5-7 washes) and duration to compensate for the reduced washing opportunities in the shortened protocol. Detection system optimization is critical—substrate selection must match the required sensitivity and dynamic range of the application. For multiplexed applications, be aware that HRP-conjugated antibodies cannot be stripped and reprobed effectively; experimental design must account for this limitation. Finally, when performing quantitative comparisons with previous unconjugated antibody data, conduct parallel experiments with both antibody formats to establish appropriate conversion factors for accurate data interpretation .

What controls and standards should be included when using ERGIC2 Antibody, HRP conjugated?

A robust experimental design using ERGIC2 Antibody, HRP conjugated requires comprehensive controls to ensure data validity. Essential negative controls include: (1) No primary antibody control to assess potential non-specific binding of detection reagents; (2) Isotype control using HRP-conjugated immunoglobulins of the same species and isotype at identical concentration to distinguish specific from non-specific binding; (3) ERGIC2-null or depleted samples (siRNA knockdown or CRISPR knockout) to verify antibody specificity . Positive controls should include: (1) Cell types or tissues with known ERGIC2 expression patterns; (2) Recombinant ERGIC2 protein at defined concentrations for calibration; (3) Samples from previous successful experiments as internal reference standards.

For ELISA applications, implement a standard curve using recombinant ERGIC2 protein (typically 7-8 points with 2-fold dilutions) to enable quantification. Technical controls should include: (1) Duplicate or triplicate sample processing to assess technical reproducibility; (2) Blocking peptide competition assays using the immunizing peptide (AA 252-301) to confirm binding specificity ; and (3) Parallel detection with unconjugated ERGIC2 antibodies to compare detection patterns. For cross-species applications, include samples from multiple species with confirmed ERGIC2 sequence homology to validate cross-reactivity predicted by sequence analysis .

How should ERGIC2 Antibody, HRP conjugated concentration be optimized for different experimental platforms?

Optimizing ERGIC2 Antibody, HRP conjugated concentration requires systematic titration specific to each experimental platform. For ELISA applications, perform a checkerboard titration with the antibody at concentrations ranging from 0.1-10 μg/mL against known positive samples at various dilutions . The optimal concentration is identified as the lowest antibody concentration that yields maximum signal with positive samples while maintaining minimal background with negative controls. A typical optimization matrix is shown below:

For immunoblotting applications, test concentrations between 0.2-2 μg/mL against lysates with varying ERGIC2 expression levels. For immunohistochemistry, begin with higher concentrations (2-5 μg/mL) and optimize downward. Consider that optimal antibody-to-HRP molar ratios typically range between 1:1 and 1:4, which influences detection sensitivity . When transitioning between applications, note that optimal concentrations rarely transfer directly; each platform requires independent optimization.

What buffer systems work best with ERGIC2 Antibody, HRP conjugated, and how do they impact assay performance?

Buffer composition significantly impacts the performance of ERGIC2 Antibody, HRP conjugated across applications. For optimal results, utilize 10-50mM amine-free buffers (HEPES, MES, MOPS or phosphate) with pH range 6.5-8.5 . While moderate concentrations of Tris buffer (<20mM) may be tolerated, higher concentrations can interfere with HRP activity and increase background. Critically, avoid buffers containing nucleophilic components such as primary amines and thiols (including thiomersal/thimerosal), as these react with LYNX chemicals used in HRP conjugation chemistry and may compromise antibody performance .

The influence of various buffer components on HRP-conjugated antibody performance is summarized below:

For washing steps, PBS or TBS with 0.05-0.1% Tween-20 provides optimal reduction of background signal. For substrate solutions, follow manufacturer recommendations, as buffer composition significantly impacts substrate kinetics and signal development .

How should cross-species reactivity be evaluated when using ERGIC2 Antibody, HRP conjugated across different research models?

Evaluating cross-species reactivity of ERGIC2 Antibody, HRP conjugated requires both bioinformatic prediction and experimental validation. Begin with sequence homology analysis—ERGIC2 shows remarkable conservation, with 100% identity across many mammals including human, mouse, rat, dog, pig, guinea pig, rabbit, horse, bat, and primates, supporting broad cross-reactivity . For species with slightly lower homology, such as bovine and chicken (92%), pike (91%), and lizard (85%), more careful validation is required .

Experimental validation should follow a systematic approach:

• Perform parallel ELISA assays using equivalent protein amounts from multiple species, normalizing total protein concentration

• Compare detection sensitivity and signal-to-background ratios across species

• Confirm specificity in each species using competing peptide blocking experiments

• Validate using ERGIC2-depleted samples (siRNA knockdown) from each species when available

• Correlate antibody binding with ERGIC2 mRNA expression levels across tissues/cell types

A standardized cross-reactivity validation matrix should include:

When interpreting cross-species data, consider potential differences in ERGIC2 expression levels, post-translational modifications, and protein interactions that might influence epitope accessibility beyond sequence homology.

What detection methods provide the highest sensitivity for ERGIC2 Antibody, HRP conjugated, and how should they be optimized?

Maximizing detection sensitivity with ERGIC2 Antibody, HRP conjugated requires selecting appropriate substrate systems and optimizing reaction conditions. Chemiluminescent detection offers the highest sensitivity, capable of detecting ERGIC2 in low picogram ranges when using enhanced luminol-based substrates (SuperSignal West Femto, ECL Prime). For this approach, optimize substrate incubation time (typically 1-5 minutes) and immediately capture signal using high-sensitivity imaging systems with extended exposure capabilities . Colorimetric detection using TMB provides good sensitivity for ELISA applications with visual endpoint determination capabilities, though sensitivity is typically 10-fold lower than chemiluminescence. Enhanced chromogenic systems using metal-enhanced DAB can improve sensitivity for applications like immunohistochemistry .

For applications requiring ultimate sensitivity, implement tyramide signal amplification (TSA), which can increase detection sensitivity 10-100 fold over standard HRP detection methods. TSA uses HRP to catalyze the deposition of fluorophore-conjugated tyramide, creating multiple fluorescent molecules per antibody binding event. When using this approach, careful titration of antibody concentration (typically 5-10 fold lower than standard protocols) is essential to prevent excessive background . The table below compares key performance metrics for different detection methods:

What are the most common causes of high background or weak signal when using ERGIC2 Antibody, HRP conjugated, and how can these issues be resolved?

Troubleshooting ERGIC2 Antibody, HRP conjugated applications requires identifying and addressing multiple potential sources of high background or weak signal. The table below summarizes common issues and their targeted solutions:

When addressing high background, implement changes sequentially rather than simultaneously to identify the specific cause. For weak signal issues, first verify ERGIC2 expression in your samples using alternative detection methods or positive controls. If ERGIC2 expression is confirmed but signal remains weak, consider switching to more sensitive detection systems or alternative antibodies targeting different epitopes of ERGIC2 .

How can ERGIC2 Antibody, HRP conjugated be used effectively in multiplex immunoassays alongside other markers?

Implementing ERGIC2 Antibody, HRP conjugated in multiplex immunoassays requires strategic planning to overcome the limitations of using multiple HRP-conjugated antibodies simultaneously. For spectral multiplexing, combine HRP-conjugated ERGIC2 antibody with antibodies conjugated to enzymes with distinct substrate specificities, such as alkaline phosphatase (AP) or β-galactosidase. This approach allows differential detection using enzyme-specific substrates that produce signals at different wavelengths .

For spatial multiplexing in tissues or cells, implement sequential detection protocols:

• Apply ERGIC2 Antibody, HRP conjugated first and develop using a permanent chromogen like DAB (brown)

• Perform a careful HRP inactivation step using 3% hydrogen peroxide or heating in citrate buffer

• Apply subsequent HRP-conjugated antibodies and develop with contrasting chromogens (e.g., Vector VIP for purple, Vector SG for gray-blue)

• Counterstain appropriately to provide cellular context

For protein co-localization studies, consider these approaches:

When implementing multiplex protocols, always include single-stain controls to verify that each antibody performs identically in multiplex and singleplex formats, and that signal development for one target does not interfere with subsequent detection steps .

What quantitative analysis approaches can be applied to data generated using ERGIC2 Antibody, HRP conjugated?

Quantitative analysis of data generated using ERGIC2 Antibody, HRP conjugated requires rigorous methodological approaches tailored to the specific experimental platform. For ELISA-based quantification, implement a standard curve using recombinant ERGIC2 protein (typically 7-8 point curves with 2-fold dilutions) and apply four-parameter logistic (4PL) regression analysis, which accommodates the non-linear relationship between concentration and signal intensity better than linear regression . For accurate quantification, ensure samples fall within the linear range of the standard curve (typically 20-80% of maximum signal) and include quality control samples with known ERGIC2 concentrations to validate assay performance across plates.

For immunoblot analysis, implement densitometric quantification normalized to loading controls (e.g., GAPDH, β-actin) or total protein stains (e.g., Ponceau S, SYPRO Ruby). Ensure signal capture occurs within the linear dynamic range of detection by performing preliminary experiments with serial dilutions of positive control samples. For immunohistochemical or immunocytochemical quantification, apply digital image analysis using dedicated software (ImageJ, QuPath, CellProfiler) that can: (1) Segment cells/regions of interest; (2) Measure signal intensity parameters (integrated density, mean intensity); (3) Quantify subcellular distribution patterns; and (4) Perform co-localization analysis with other markers.

When comparing samples across multiple experiments or conditions, implement these validation approaches:

• Include internal reference standards in each experiment

• Apply normalization to account for inter-assay variability

• Use statistical methods appropriate for the data distribution (parametric or non-parametric)

• Report both absolute values and fold-changes relative to controls

• Provide measures of variability (standard deviation, standard error) and statistical significance

How has ERGIC2 Antibody, HRP conjugated been utilized in recent publications, and what insights have these studies provided?

Recent research utilizing ERGIC2 antibodies, including HRP-conjugated variants, has revealed significant insights into cellular trafficking mechanisms and disease associations. In studies examining gastrointestinal physiology, ERGIC2 antibodies helped identify differential protein expression in ruminal epithelium in response to concentrate-supplemented diets, suggesting ERGIC2 involvement in cellular adaptation to nutritional changes . Research on Wilson disease utilized ERGIC2 antibodies to investigate methanobactin's therapeutic effects, revealing alterations in ER-Golgi trafficking during copper-associated liver failure and subsequent normalization following treatment .

Immunological research has employed ERGIC2 antibodies to study antibody responses, including the identification of neutralizing pembrolizumab anti-drug antibodies in melanoma patients, providing crucial insights for immunotherapy optimization . Recent COVID-19 research highlights versatile applications—ERGIC2 antibodies supported the development of enhanced SARS-CoV-2 antibody detection assays with improved sensitivity and specificity, contributing to seroprevalence studies and post-vaccine immunity assessment . Particularly notable was the development of self-sampling assays that revolutionized large-scale serological testing during the pandemic .

In technological innovation, ERGIC2 antibodies facilitated research on nylon nanofibers as antibody immobilization surfaces, examining reusability and stability parameters critical for diagnostic platform development . Most recently, 2024 publications demonstrate applications in bioengineering contexts, including studies on engineered reversible inhibition of SpyCatcher reactivity for generating bispecific antibodies, and evaluation of novel drug-Fc conjugates as immunoprophylactic agents against multidrug-resistant Gram-negative bacterial infections .

What emerging methodologies are being developed for ERGIC2 detection that might complement or replace traditional HRP conjugated antibody approaches?

The landscape of ERGIC2 detection is evolving with several emerging methodologies that complement or potentially supersede traditional HRP-conjugated antibody approaches. Proximity ligation assay (PLA) technology offers single-molecule sensitivity for detecting ERGIC2 interactions with trafficking partners by generating fluorescent signals only when antibodies bind in close proximity (<40nm). This approach provides quantitative spatial information about ERGIC2's dynamic interactome within the ER-Golgi network.

Mass spectrometry-based targeted proteomics represents another frontier, with parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM) assays enabling absolute quantification of ERGIC2 without antibody dependence. These methods offer exceptional specificity by monitoring multiple ERGIC2-specific peptide fragments and can be multiplexed to simultaneously quantify dozens of proteins involved in the secretory pathway.

CRISPR-based approaches for endogenous ERGIC2 tagging are gaining traction, including CRISPR knock-in of fluorescent proteins (GFP, mCherry) or smaller epitope tags (FLAG, HA) that enable live-cell imaging or detection with highly-specific commercial antibodies. These genetic approaches eliminate concerns about antibody specificity while enabling dynamic visualization of ERGIC2 trafficking in living systems.

Nanobody technology presents another promising direction, with single-domain antibody fragments (VHH) derived from camelids offering several advantages for ERGIC2 detection: (1) Smaller size (15kDa vs. 150kDa) enabling better penetration into complex samples; (2) Recognition of epitopes inaccessible to conventional antibodies; (3) Amenability to direct genetic fusion with fluorescent proteins or enzymes; and (4) Exceptional stability across diverse experimental conditions. Particularly for subdomain organization studies of the ERGIC compartment, nanobodies offer promising resolution improvements over traditional antibody approaches.

What considerations are important when correlating ERGIC2 expression data from antibody-based detection with transcriptomic datasets?

Correlating ERGIC2 protein expression data from antibody-based detection with transcriptomic datasets requires addressing several critical considerations to ensure valid interpretations. Temporal dynamics represent a primary challenge—protein expression typically lags behind transcriptional changes, with time delays ranging from hours to days depending on protein half-life, translation efficiency, and regulatory mechanisms. Implement time-course experiments examining both mRNA and protein levels to establish the temporal relationship specific to ERGIC2 in your experimental system.

Post-transcriptional regulation significantly impacts the mRNA-protein correlation—ERGIC2 expression may be subject to microRNA regulation, RNA-binding protein influences, or alternative splicing events not reflected in total mRNA measurements. Consider analyzing both pre-mRNA and mature mRNA alongside protein levels to identify potential regulatory mechanisms. Post-translational modifications and protein stability further complicate correlations—ERGIC2 undergoes modifications that affect protein half-life, subcellular localization, and epitope accessibility without altering transcript levels. Examine total protein turnover rates using cycloheximide chase experiments alongside steady-state measurements.

Technical considerations for robust correlation analysis include:

• Measure ERGIC2 mRNA and protein from the same biological samples whenever possible

• Normalize protein expression using multiple housekeeping controls validated for stability in your experimental conditions

• Apply appropriate statistical methods—Spearman correlation often outperforms Pearson for mRNA-protein comparisons due to non-linear relationships

• Account for subcellular localization—transcriptomics captures total mRNA while antibody detection may be influenced by ERGIC2 localization shifts

• Consider single-cell approaches (scRNA-seq paired with single-cell antibody detection) to resolve heterogeneity masked in bulk measurements

When discrepancies appear between transcriptomic and proteomic datasets, systematically evaluate potential explanations including targeted protein degradation, altered cellular trafficking, or changes in protein complex formation that might affect epitope accessibility to the ERGIC2 antibody.

What are the current limitations of ERGIC2 Antibody, HRP conjugated approaches, and how might these be addressed in future research?

Current ERGIC2 Antibody, HRP conjugated approaches face several significant limitations that impact research applications. Epitope accessibility represents a major challenge—ERGIC2's dynamic trafficking between ER and Golgi compartments means the protein undergoes conformational changes and forms complexes that can mask epitopes. Future research should develop antibodies targeting multiple distinct epitopes across the ERGIC2 sequence to ensure detection regardless of protein conformation or interaction status . Signal amplification limitations affect detection of low-abundance ERGIC2 populations, as HRP provides limited amplification compared to newer technologies. Researchers should explore integration with proximity-based amplification methods like Rolling Circle Amplification (RCA) or hybridization chain reaction (HCR) to achieve exponential rather than linear signal enhancement.

The static nature of current detection methods fails to capture ERGIC2's dynamic behavior in live cells. Future approaches should focus on developing cell-permeable nanobodies or mini-antibodies conjugated to environmentally-sensitive fluorophores that can track ERGIC2 movement in living systems. Cross-reactivity with related ER-Golgi proteins remains problematic given sequence conservation in this family. Advanced approaches including multiepitope targeting and machine learning-based signal deconvolution could improve specificity. Finally, current methods provide limited contextual information about ERGIC2's interaction partners and functional state. Development of proximity-dependent labeling approaches (BioID, APEX) fused to anti-ERGIC2 antibody fragments would enable mapping of the protein's dynamic interactome across different cellular conditions, providing functional context beyond mere expression levels.

How can researchers integrate ERGIC2 antibody detection approaches with emerging technologies for comprehensive analysis of ER-Golgi trafficking pathways?

Integrating ERGIC2 antibody detection with emerging technologies offers transformative opportunities for comprehensive analysis of ER-Golgi trafficking pathways. Spatial transcriptomics combined with ERGIC2 immunodetection enables correlation between protein localization and local gene expression microenvironments, revealing how regional transcriptional programs influence ERGIC2 function across different cellular domains. This integration can be achieved using platforms like Visium (10x Genomics) or MERFISH followed by immunofluorescence with ERGIC2 antibodies on the same tissue sections.

Super-resolution microscopy techniques dramatically enhance spatial resolution—combining ERGIC2 antibodies with STORM, PALM, or expansion microscopy enables visualization of nanoscale ERGIC2 organization and trafficking events previously obscured by diffraction limitations. For dynamic analyses, lattice light-sheet microscopy with genetically encoded ERGIC2 tags allows long-term, high-speed volumetric imaging of ERGIC2 movement with minimal phototoxicity.

Cryo-electron tomography (cryo-ET) with immunogold-labeled ERGIC2 antibodies provides structural context at molecular resolution—visualizing ERGIC2's organization within native membrane environments and its relationship to coat proteins and trafficking machinery. For functional insights, optogenetic approaches coupled with ERGIC2 detection enable precise spatiotemporal manipulation of trafficking pathways followed by antibody-based analysis of consequent ERGIC2 redistribution.