ergic3 Antibody

What is ERGIC3 and what cellular compartments express this protein?

ERGIC3 is located in the cis face of the Golgi apparatus and vesicular tubular structures between the transitional endoplasmic reticulum (ER) and cis-Golgi . Functionally, it plays a role in transport between the endoplasmic reticulum and Golgi, positively regulating trafficking of secretory proteins like SERPINA1/alpha-1-antitrypsin and HP/haptoglobin . In normal human tissues, ERGIC3 expression is limited to certain epithelial cells, including hepatocytes, gastrointestinal epithelium, ducts and acini of the pancreas, proximal and distal tubules of the kidney, and mammary epithelial cells, while most normal human tissues do not express it .

What are the recommended applications for ERGIC3 antibodies in laboratory research?

ERGIC3 antibodies have been validated for multiple applications:

It is recommended that researchers titrate the antibody in each specific testing system to obtain optimal results, as sample-dependent variations may occur .

How should researchers optimize storage conditions for ERGIC3 antibodies?

Most ERGIC3 antibodies should be stored at -20°C and are typically stable for one year after shipment . The storage buffer usually contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. Aliquoting is recommended to avoid repeated freeze/thaw cycles which can compromise antibody performance . Some preparations (20μl sizes) may contain 0.1% BSA, which should be noted when designing experiments .

What controls should be included when using ERGIC3 antibodies for Western blot analysis?

For rigorous Western blot analysis with ERGIC3 antibodies:

• Include positive controls: HepG2 cells, K-562 cells, MCF-7 cells, HEK-293 cells, and HeLa cells have been validated as expressing ERGIC3 .

• Include negative controls: ERGIC3 knockout cell lines such as Human ERGIC3 knockout HEK-293T cell line (ab266489) with corresponding lysate (ab257941) have been used to demonstrate antibody specificity .

• Always include loading controls: GAPDH antibody (e.g., ab8245) has been used successfully in ERGIC3 research .

• Expected molecular weight: While the calculated molecular weight of ERGIC3 is approximately 43 kDa (383 amino acids), the observed molecular weight is typically around 50 kDa due to post-translational modifications .

What are the critical parameters for successful immunohistochemical staining with ERGIC3 antibodies?

For optimal immunohistochemical detection of ERGIC3:

• Tissue preparation: Use 4-μm-thick paraffin sections from formalin-fixed tissues .

• Antigen retrieval: Heat-induced epitope retrieval in Tris-EDTA Buffer (10 mM Tris Base, 1 mM EDTA Solution, 0.05% Tween 20, pH 9.0) is recommended, though citrate buffer (pH 6.0) can be used as an alternative .

• Endogenous peroxidase elimination: Incubate sections in 3% H₂O₂ for 10 minutes .

• Antibody incubation: Follow the recommended dilution ranges (typically 1:50-1:500 for IHC) .

• Color development: 3,3'-Diaminobenzidin works effectively, with hematoxylin counterstaining for nuclei .

• Interpretation: Only manifest cytoplasmic staining should be defined as a positive reaction .

How can ERGIC3 antibodies be utilized for cancer biomarker research?

ERGIC3 shows significant potential as a cancer biomarker, particularly for non-small cell lung cancer (NSCLC):

What methodological approaches are recommended for studying interactions between ERGIC3 and other proteins?

To investigate ERGIC3 protein interactions:

• Immunoprecipitation followed by mass spectrometry: This approach has successfully identified ERGIC3-interacting proteins in both intracellular and extracellular compartments. Use isobaric tags for relative and absolute quantitation (iTRAQ) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) .

• Co-immunofluorescence studies: Combine anti-ERGIC3 antibodies with antibodies against potential interacting partners (e.g., MUC1, ST, calreticulin, and 58K Golgi protein) using FITC-coupled anti-rabbit antibody and Cy3-coupled anti-mouse antibody for visualization under confocal laser scanning microscopy .

• Differential expression analysis: After ERGIC3 knockdown, analyze changes in protein expression patterns to identify functionally related proteins. In lung cancer cells, 88 extracellular differentially expressed proteins (41 up-regulated and 47 down-regulated) and 52 intracellular differentially expressed proteins (33 up-regulated and 19 down-regulated) have been identified .

How can researchers effectively achieve and validate ERGIC3 knockdown for functional studies?

For ERGIC3 knockdown studies:

• RNAi technique: Transfect cells with ERGIC3 siRNA using Lipofectamine 3000 or similar transfection reagents. Include a negative control siRNA group .

• ERGIC3 siRNA design: Previously successful siRNA designs have targeted specific regions of the ERGIC3 mRNA. Commercial options from suppliers such as Thermo Fisher Scientific have been validated .

• Verification of knockdown: Validate knockdown efficiency using both:

  • Quantitative real-time PCR (qRT-PCR) to confirm mRNA reduction

  • Western blot with ERGIC3 antibodies to confirm protein reduction
    Effective knockdown typically reduces ERGIC3 expression to approximately 10% of control levels .

• Collection timing: Culture transfected cells for approximately 72 hours before collecting samples for subsequent experiments .

What approaches should be used to investigate the relationship between ERGIC3 and miRNA regulation?

To study miRNA-based regulation of ERGIC3:

• Bioinformatic prediction: Use algorithms to predict miRNAs that might target ERGIC3 mRNA. Previous research identified 398 potential miRNA binding sites .

• MiRNA expression profiling: Compare miRNA profiles between cancer cells and normal controls to identify differentially expressed miRNAs. Integration with bioinformatic predictions can identify candidate regulatory miRNAs .

• Validation techniques:

  • Luciferase reporter assay to confirm direct miRNA targeting

  • qRT-PCR to quantify miRNA expression levels

  • Transfection of candidate miRNAs to observe effects on ERGIC3 expression

• Key findings to build upon: miR-203a has been identified as a negative regulator of ERGIC3 in NSCLC cells, with miR-203a downregulation inducing ERGIC3 overexpression .

How can ERGIC3 antibodies be employed to investigate ERGIC3's role in immune responses?

Recent research has identified ERGIC3 as a novel immune function-related gene:

• Immunoinfiltrate correlation analysis: Use TIMER (http://timer.cistrome.org/) to analyze associations between ERGIC3 expression and the abundance of tumor immune infiltrates, including B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and myeloid dendritic cells .

• Pathway analysis: After ERGIC3 knockdown, perform transcriptomic analysis to identify differentially expressed genes involved in immune-related pathways. Previous research has shown that genes affected by ERGIC3 knockdown are enriched in vesicular transport, growth factors, PI3K-Akt, NOD-like, Jak-STAT, NF-kappa B and other protein kinase-coupled receptor-mediated signal transduction pathways related to tumor immune response .

• Extracellular protein analysis: Investigate changes in extracellular proteins after ERGIC3 knockdown, as these may mediate immune interactions. Cluster analysis has shown that extracellular differential proteins are mainly involved in Ca²⁺ binding and transport and I-kappa B kinase/NF-kappa B signal transduction .

What strategies can address weak or inconsistent signals when using ERGIC3 antibodies?

When experiencing weak or inconsistent signals:

• Antibody titration: Optimize antibody concentration by testing a range of dilutions. For Western blot, test from 1:2000 to 1:10000; for IHC-P, test from 1:50 to 1:500 .

• Antigen retrieval optimization: Try both:

  • TE buffer (pH 9.0) - primary recommendation

  • Citrate buffer (pH 6.0) - alternative method

• Sample selection: Ensure appropriate positive controls (HepG2, K-562, MCF-7 cells) are included .

• Storage assessment: Verify antibody has been stored properly at -20°C and has not undergone multiple freeze-thaw cycles .

• Detection system sensitivity: Consider switching to a more sensitive detection system, especially for tissues with lower ERGIC3 expression.

How can researchers address potential cross-reactivity issues when using ERGIC3 antibodies?

To minimize and address cross-reactivity:

• Antibody selection: Choose antibodies validated against knockout controls. For example, ab129179 Anti-ERGIC3 antibody [EPR8141] has been validated against ERGIC3 knockout HEK-293T cell line .

• Blocking optimization: Increase blocking time or concentration to reduce non-specific binding.

• Negative tissue controls: Include tissues known to be negative for ERGIC3 expression. Most normal human tissues do not express ERGIC3, with normal lung tissues being completely negative .

• Reactivity assessment: Review the tested reactivity of each antibody. Some ERGIC3 antibodies have been validated for human, mouse, and rat samples, while others are human-specific or have predicted cross-reactivity with other species .

• Epitope consideration: Select antibodies targeting unique regions of ERGIC3. Some target N-terminal regions (amino acids 1-30) , while others target internal epitopes.

What emerging applications of ERGIC3 antibodies warrant further investigation?

Based on current research trajectories:

• Liquid biopsy development: Investigate whether ERGIC3 can be detected in patient blood samples as a non-invasive diagnostic tool for early cancer detection, building on findings that ERGIC3 is strongly overexpressed in multiple carcinomas .

• Therapeutic targeting: Explore whether ERGIC3 antibodies can be developed for targeted therapy approaches, particularly for NSCLC where ERGIC3 is consistently overexpressed but absent in normal lung tissue .

• Immune checkpoint interactions: Further investigate the relationship between ERGIC3 and tumor immune infiltrates, exploring whether ERGIC3 modulates immune checkpoint molecules .

• Role in protein trafficking disorders: Study ERGIC3's function in regulating secretory protein trafficking (SERPINA1/alpha1-antitrypsin and HP/haptoglobin) and potential implications for diseases involving protein trafficking defects .

How might multiomics approaches enhance our understanding of ERGIC3 function using antibody-based techniques?

Integrative approaches for ERGIC3 research:

• Antibody-based proteomics combined with transcriptomics: Correlate ERGIC3 protein expression (detected by antibodies) with gene expression patterns to identify regulatory networks. Previous research has identified miR-203a as a regulator of ERGIC3 through such integrated approaches .

• Spatial proteomics: Combine ERGIC3 antibody staining with multiplexed imaging to understand ERGIC3's spatial relationship with other proteins in the secretory pathway and cancer microenvironment.

• Interactome mapping: Use ERGIC3 antibodies for immunoprecipitation followed by mass spectrometry to expand our understanding of ERGIC3's protein interaction network. Previous research has identified differential protein expression after ERGIC3 knockdown, suggesting complex interaction networks .

• Single-cell analysis: Apply ERGIC3 antibodies in single-cell proteomics to understand heterogeneity in ERGIC3 expression within tumors and correlate with cellular phenotypes and functions.