SEC23IP Antibody, HRP conjugated

What is SEC23IP and what role does it play in cellular processes?

SEC23IP (SEC23-Interacting Protein) plays a critical role in the organization of endoplasmic reticulum exit sites (ERES). The protein specifically binds to phosphatidylinositol 3-phosphate (PI(3)P), phosphatidylinositol 4-phosphate (PI(4)P), and phosphatidylinositol 5-phosphate (PI(5)P) . SEC23IP forms distinguishable puncta throughout the cytosol that colocalize with Sec31A, a well-established ERES marker, with a substantial fraction of these puncta tightly associated with the cis-/medial Golgi apparatus .

SEC23IP was originally identified through its interaction with proteins similar to yeast Sec23p, which is an essential component of COPII-coated vesicles involved in protein transport from the endoplasmic reticulum to the Golgi apparatus . Recent research has demonstrated that SEC23IP shares significant similarity with phospholipid-modifying proteins, particularly phosphatidic acid preferring-phospholipase A1 . Of particular interest to cellular biologists, overexpression of SEC23IP has been shown to cause disorganization of the endoplasmic reticulum-Golgi intermediate compartment and Golgi apparatus, suggesting its critical role in maintaining cellular compartment integrity .

What applications is SEC23IP Antibody (HRP) most suitable for in experimental protocols?

The SEC23IP Antibody (HRP) is primarily validated for ELISA applications, as indicated in the product specifications . This antibody is particularly valuable for researchers investigating protein transport pathways, ER-Golgi trafficking, and cellular compartment organization. The HRP conjugation provides significant advantages for detection sensitivity in immunoassays through enzymatic signal amplification.

For ELISA applications, researchers should determine optimal dilutions experimentally, as these may vary depending on the specific experimental conditions and detection systems employed . The high purity (>95%) of this polyclonal antibody, combined with its HRP conjugation, makes it particularly suitable for quantitative detection of SEC23IP in human samples .

While ELISA is the primary validated application, researchers may extend its use to other immunological techniques that benefit from HRP detection systems, though additional validation would be necessary for these applications.

What are the optimal storage and handling conditions for maintaining SEC23IP Antibody (HRP) activity?

To maintain optimal activity of SEC23IP Antibody (HRP), proper storage and handling are essential. The antibody should be aliquoted and stored at -20°C to prevent repeated freeze-thaw cycles which can degrade antibody performance . It is particularly important to avoid exposure to light as the HRP conjugate is photosensitive .

The antibody is supplied in liquid form containing 0.01 M PBS (pH 7.4), 0.03% Proclin-300, and 50% glycerol . This buffer formulation helps maintain stability during storage. When working with the antibody, it should be thawed completely and mixed gently before use to ensure homogeneity.

For long-term storage integrity, consider the following practices:

• Create multiple small-volume aliquots immediately upon receipt

• Store in amber tubes or wrapped in foil to prevent light exposure

• Avoid repeated freeze-thaw cycles (more than 3-5 cycles can significantly reduce activity)

• Allow the antibody to equilibrate to room temperature before opening stored aliquots

How does HRP conjugation affect antibody performance compared to unconjugated antibodies?

HRP (Horseradish Peroxidase) conjugation significantly enhances detection sensitivity in immunoassay applications compared to unconjugated antibodies. The enzymatic activity of HRP provides signal amplification by catalyzing the oxidation of substrates to produce colorimetric, chemiluminescent, or fluorescent signals, depending on the detection system used .

Research has demonstrated that the method of HRP conjugation to antibodies can dramatically impact performance. Traditional conjugation methods can result in varying enzyme-to-antibody ratios, but modified protocols incorporating a lyophilization step have shown enhanced binding capacity and superior performance . In comparative studies, HRP-conjugated antibodies prepared with lyophilization worked effectively at dilutions of 1:5000, whereas traditionally prepared conjugates required much higher concentrations (1:25 dilution), representing a 200-fold improvement in sensitivity (p < 0.001) .

The conjugation process must balance maximum enzyme loading without compromising antibody binding activity. The chemical modification typically uses sodium meta periodate to generate aldehyde groups by oxidizing carbohydrate moieties on the HRP molecule, which then form covalent bonds with primary amines on the antibody .

What is the molecular mechanism of SEC23IP interaction with VPS13B at the ER exit site-Golgi interface?

The interaction between SEC23IP and VPS13B (also known as COH1) represents a critical molecular mechanism at the ER exit site-Golgi interface. Recent research has identified SEC23IP as a VPS13B adaptor that recruits VPS13B to ERES-Golgi interfaces . This interaction was confirmed through co-immunoprecipitation and mass spectrometry, demonstrating that endogenous SEC23IP can be copelleted with endogenous VPS13B .

The molecular interaction involves specific domains: VPS13B binds to SEC23IP through its VAB (VPS13A/C Binding) domain . Further structural analysis revealed that this VAB domain contains six repeats (R1-R6), with repeats R2-R5 acting as the core complex for binding to SEC23IP, while R1 and R6 provide supplementary interaction surfaces . Notably, in vitro pulldown assays confirmed that the VAB domain directly binds to SEC23IP, with regions containing R1-R5 showing significantly stronger binding than those containing only R1-R4 .

The functional consequence of this interaction is dramatic recruitment of ERES to the Golgi apparatus. When VPS13B and SEC23IP are co-expressed, a substantially larger proportion of SEC23IP puncta are recruited to the Golgi, creating an extensive ERES-Golgi interface with most ERES tightly associated with the cis-Golgi . This recruitment is so extensive that it results in significantly fewer ERES in the cell periphery, emphasizing the strength of this molecular interaction .

How does SEC23IP contribute to Golgi structure maintenance and what happens when it is disrupted?

SEC23IP plays a crucial role in maintaining Golgi structure and organization. CRISPR-Cas9-mediated knockout of SEC23IP results in fragmentation of the Golgi apparatus, phenocopying the effect observed in VPS13B knockout cells . This suggests that SEC23IP works in conjunction with VPS13B to maintain Golgi integrity.

The mechanistic basis for this maintenance involves SEC23IP's ability to form puncta that colocalize with Sec31A (an ERES marker) and associate with the cis-/medial Golgi . Through its interaction with VPS13B, SEC23IP facilitates the formation of ERES-Golgi interfaces, which are essential for proper vesicular trafficking between these compartments .

When the SEC23IP-VPS13B interaction is disrupted, several cellular consequences are observed:

• Fragmentation of the Golgi apparatus

• Altered distribution of ERES throughout the cell

• Disrupted vesicular trafficking between the ER and Golgi

• Potential downstream effects on protein secretion and glycosylation

This relationship explains why disruptions to either SEC23IP or VPS13B can lead to similar cellular phenotypes, as they function in the same pathway to maintain Golgi structure and function.

What methodological considerations are important when optimizing SEC23IP Antibody (HRP) for immunoassays?

When optimizing SEC23IP Antibody (HRP) for immunoassays such as ELISA, several methodological considerations must be addressed to ensure maximum sensitivity and specificity:

Antibody Dilution Optimization:
Researchers should perform systematic dilution series to identify the optimal antibody concentration that provides maximum signal-to-noise ratio. While the product specifications indicate that optimal dilutions should be determined by the end user , starting with a range of 1:1000 to 1:10,000 is recommended based on the high sensitivity of HRP-conjugated antibodies prepared with advanced conjugation methods .

Blocking Conditions:
Proper blocking is critical to minimize non-specific binding. Consider testing multiple blocking agents (BSA, casein, non-fat dry milk) at different concentrations to identify optimal conditions for your specific assay system.

HRP Substrate Selection:
The choice of substrate significantly impacts sensitivity. Options include:

• TMB (3,3',5,5'-Tetramethylbenzidine): High sensitivity, colorimetric detection

• ECL (Enhanced Chemiluminescence): Superior sensitivity for chemiluminescent detection

• ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)): Lower sensitivity but stable color development

Assay Buffer Composition:
Buffer composition should be optimized to maintain both antibody binding efficiency and HRP enzymatic activity. Consider testing different pH ranges (7.0-7.5) and including stabilizers such as BSA (0.1-0.5%) to improve performance.

Temperature and Incubation Time:
Systematic optimization of both temperature and incubation time is recommended. While room temperature incubation (20-25°C) for 1-2 hours is standard, some assays may benefit from longer incubation at 4°C or shorter incubation at 37°C.

How can the effect of lyophilization be leveraged to enhance SEC23IP Antibody-HRP conjugation efficiency?

Lyophilization can significantly enhance SEC23IP Antibody-HRP conjugation efficiency, as demonstrated by studies on HRP-antibody conjugation methodologies . Incorporating a lyophilization step into the conjugation protocol has been shown to dramatically improve the binding capacity of antibodies to HRP molecules.

The enhanced conjugation process typically follows these steps:

• Activation of HRP using sodium meta periodate to generate aldehyde groups through oxidation of carbohydrate moieties

• Lyophilization of the activated HRP (this is the critical additional step)

• Reconstitution and mixing with antibody at appropriate concentration (typically 1 mg/ml)

• Reduction of Schiff bases using sodium cyanoborohydride

• Purification of the conjugate

This modified protocol has demonstrated remarkable improvements in conjugate performance, with conjugates prepared using lyophilization working effectively at dilutions as high as 1:5000, compared to traditionally prepared conjugates requiring much higher concentrations (1:25 dilution) . This represents a 200-fold improvement in sensitivity with high statistical significance (p < 0.001) .

The mechanism behind this enhancement likely involves better spatial arrangement and increased accessibility of aldehyde groups on the HRP molecule after lyophilization, allowing more efficient coupling with primary amines on the antibody. Additionally, the removal of water during lyophilization may promote more favorable reaction conditions by increasing local concentrations of reactants.

What is the relationship between SEC23IP and Cohen syndrome through its interaction with VPS13B/COH1?

The relationship between SEC23IP and Cohen syndrome is established through SEC23IP's direct interaction with VPS13B (also known as COH1), mutations in which cause Cohen syndrome . Cohen syndrome is a rare autosomal recessive disorder characterized by developmental delay, microcephaly, hypotonia, retinal dystrophy, and distinctive facial features.

Recent research has elucidated that SEC23IP serves as an adaptor protein for VPS13B, recruiting it to the ER exit site-Golgi interface . This interaction is critical for maintaining proper Golgi structure and function. Several lines of evidence support this relationship:

• Both SEC23IP and VPS13B knockout result in similar cellular phenotypes, particularly Golgi fragmentation

• SEC23IP directly interacts with VPS13B through specific domain interactions (the VAB domain of VPS13B with SEC23IP)

• This interaction facilitates extensive recruitment of ERES to the Golgi, creating an ERES-Golgi interface that is essential for proper vesicular trafficking

The molecular mechanism involves SEC23IP at ERES binding to VPS13B at the Golgi membrane, essentially bridging these two cellular compartments . When either protein is absent or dysfunctional, this bridge fails to form properly, leading to Golgi fragmentation and disrupted trafficking - cellular defects that likely contribute to Cohen syndrome pathophysiology.

Interestingly, both VPS13B and SEC23IP are vertebrate-specific proteins with no homologs in yeast or Caenorhabditis elegans, suggesting they evolved to support specialized functions in higher organisms . This evolutionary specificity may partly explain why disruptions to this pathway result in the complex developmental and neurological features characteristic of Cohen syndrome.

What analytical techniques can be combined with SEC23IP Antibody (HRP) for comprehensive cellular localization studies?

For comprehensive cellular localization studies using SEC23IP Antibody (HRP), several complementary analytical techniques can be employed:

Immunofluorescence (IF) with Confocal Microscopy:
While the SEC23IP Antibody (HRP) is primarily designed for ELISA applications, researchers can use unconjugated variants for IF studies or employ secondary detection systems compatible with HRP. This approach has been successfully used to demonstrate that SEC23IP forms puncta colocalizing with Sec31A (an ERES marker) with a substantial fraction associated with the cis-/medial Golgi .

Proximity Ligation Assay (PLA):
PLA can detect protein-protein interactions at endogenous levels with high specificity and sensitivity. This technique would be particularly valuable for confirming the SEC23IP-VPS13B interaction in different cell types or experimental conditions.

Correlative Light and Electron Microscopy (CLEM):
CLEM combines the benefits of fluorescence microscopy with the ultrastructural resolution of electron microscopy. This approach would provide detailed insights into the precise localization of SEC23IP at ERES and its relationship to membrane structures.

Live-Cell Imaging:
For dynamic studies, SEC23IP can be tagged with fluorescent proteins and combined with markers for different cellular compartments. This approach has been used to demonstrate the dramatic recruitment of SEC23IP to the Golgi when co-expressed with VPS13B .

Immuno-Electron Microscopy:
For ultrastructural localization studies, gold-labeled secondary antibodies can be used with the primary SEC23IP antibody to precisely map its distribution at the nanometer scale.

What controls should be included when using SEC23IP Antibody (HRP) in experimental protocols?

When using SEC23IP Antibody (HRP) in experimental protocols, several essential controls should be included to ensure result validity and interpretability:

Negative Controls:

• Isotype Control: Use a rabbit IgG (matching the SEC23IP antibody's isotype) at the same concentration to identify non-specific binding

• Secondary Antibody Only: For indirect detection methods, include wells/samples treated only with secondary antibody

• Knockout/Knockdown Validation: Where possible, include SEC23IP knockout or knockdown samples to confirm specificity

Positive Controls:

• Known Positive Sample: Include samples with confirmed SEC23IP expression

• Recombinant SEC23IP: Use purified recombinant protein as a standard reference

• Overexpression System: Cells transfected with SEC23IP expression constructs serve as high-expression positive controls

Technical Controls:

• HRP Activity Control: Include an HRP activity test to confirm enzymatic function is maintained

• Dilution Series: Perform a dilution series of both antibody and sample to establish the linear detection range

• Cross-Reactivity Assessment: Test antibody against closely related proteins if available

Experimental Design Controls:

• Biological Replicates: Include multiple biological replicates to account for natural variation

• Technical Replicates: Perform at least triplicate measurements of each sample

• Recovery Test: For quantitative assays, spike known amounts of target protein into samples to assess recovery efficiency

How can SEC23IP Antibody (HRP) be utilized to investigate ER-Golgi trafficking defects in neurodevelopmental disorders?

SEC23IP Antibody (HRP) offers valuable opportunities for investigating ER-Golgi trafficking defects in neurodevelopmental disorders, particularly Cohen syndrome, which is caused by mutations in VPS13B/COH1 . Recent discoveries revealing SEC23IP as a critical adaptor that recruits VPS13B to ER exit site-Golgi interfaces provide a mechanistic foundation for such investigations .

Researchers can employ this antibody to:

• Quantify SEC23IP Levels in Patient-Derived Cells:
  Using ELISA with SEC23IP Antibody (HRP), researchers can measure SEC23IP protein levels in patient-derived cells to determine if VPS13B mutations affect SEC23IP expression or stability.

• Assess ER-Golgi Interface Integrity:
  Since the SEC23IP-VPS13B interaction is critical for ERES-Golgi association, this antibody can be used to evaluate whether this interface is disrupted in neuronal models of Cohen syndrome or related disorders.

• Monitor Therapeutic Interventions:
  For therapeutic development, this antibody can serve as a tool to monitor whether interventions restore proper SEC23IP localization and function in cellular models of disease.

• Investigate Related Neurodevelopmental Disorders:
  Given the importance of ER-Golgi trafficking in neuronal function, this antibody could help investigate whether similar molecular mechanisms are disrupted in other neurodevelopmental disorders with Golgi fragmentation phenotypes.

• Develop High-Throughput Screening Assays:
  The HRP conjugation enables development of high-throughput screening assays to identify compounds that might restore proper SEC23IP-VPS13B interactions or downstream functions.

What insights does the SEC23IP-VPS13B interaction provide for understanding lipid transport mechanisms?

The SEC23IP-VPS13B interaction provides significant insights into lipid transport mechanisms at membrane contact sites between the ER and Golgi. This interaction represents a molecular bridge between these organelles that likely facilitates lipid exchange and membrane homeostasis.

SEC23IP has been shown to specifically bind phosphatidylinositol 3-phosphate (PI(3)P), phosphatidylinositol 4-phosphate (PI(4)P), and phosphatidylinositol 5-phosphate (PI(5)P) . This phosphoinositide-binding property suggests that SEC23IP may help position ERES at specific membrane domains enriched in these lipids. Additionally, SEC23IP shares significant similarity with phospholipid-modifying proteins, particularly phosphatidic acid preferring-phospholipase A1 , suggesting it may directly participate in lipid metabolism at these sites.

VPS13B belongs to the VPS13 family of proteins, which have been implicated in lipid transport between membranes at organelle contact sites. The direct interaction between SEC23IP and VPS13B through the VAB domain suggests that this protein complex may coordinate lipid transport at ER-Golgi interfaces.

This molecular arrangement provides a framework for understanding how lipid composition is maintained between the ER and Golgi, which is critical for proper membrane trafficking and organelle function. Disruptions to this system, as seen in Cohen syndrome, may lead to altered membrane composition and subsequently impaired function of the secretory pathway.

What are the implications of SEC23IP research for understanding cellular stress responses and protein quality control?

Research on SEC23IP has significant implications for understanding cellular stress responses and protein quality control mechanisms. As a key organizer of ERES and facilitator of ER-Golgi trafficking through its interaction with VPS13B , SEC23IP likely plays important roles in cellular adaptation to stress conditions.

During ER stress, which occurs when misfolded proteins accumulate in the ER, cells activate the unfolded protein response (UPR). This response involves expansion of the ER, upregulation of chaperones, and modulation of ER-Golgi trafficking. Given SEC23IP's role in organizing ERES , it may be involved in regulating the export of proteins from the ER during stress conditions.

The fragmentation of the Golgi observed in SEC23IP knockout cells suggests that this protein is essential for maintaining Golgi integrity. Golgi fragmentation is a common cellular response to various stressors, including oxidative stress and protein misfolding. Therefore, SEC23IP may function as a stress-responsive factor that helps maintain secretory pathway function under adverse conditions.

Additionally, the interaction between SEC23IP and VPS13B at the ER-Golgi interface may facilitate communication between these organelles during stress, allowing for coordinated responses to cellular challenges. This interaction could be regulated by post-translational modifications or changes in lipid composition that occur during stress responses.

Understanding these mechanisms could provide insights into various diseases associated with ER stress and protein quality control defects, including neurodegenerative disorders, metabolic diseases, and certain forms of cancer.

What are the complete technical specifications for SEC23IP Antibody (HRP)?

The following table presents the comprehensive technical specifications for SEC23IP Antibody (HRP) based on product information:

This antibody is specifically designed for research applications focused on SEC23IP, with high purity and HRP conjugation that enhances detection sensitivity in immunoassay applications .

How does lyophilization impact HRP-antibody conjugate performance parameters?

The impact of lyophilization on HRP-antibody conjugate performance parameters is substantial, as demonstrated by comparative studies. The following table summarizes the key performance differences between traditional conjugation methods and those incorporating a lyophilization step:

The significant improvement in effective working dilution (from 1:25 to 1:5000) represents a 200-fold increase in sensitivity with high statistical significance (p < 0.001) . This dramatic enhancement is attributed to the lyophilization step allowing antibodies to bind more HRP molecules without compromising binding specificity .

The mechanism behind this improvement involves the lyophilization process creating structural changes in the activated HRP that favor more efficient conjugation when mixed with antibodies. This modified protocol represents a significant advancement in antibody-enzyme conjugation technology that can be applied to various antibodies, including SEC23IP Antibody (HRP) .