SEC16A Antibody

What is SEC16A and what cellular functions does it mediate?

SEC16A (also known as KIAA0310, SEC16, and SEC16L) is a critical protein required for secretory cargo traffic from the endoplasmic reticulum (ER) to the Golgi apparatus. It functions as a scaffold protein at ER exit sites (ERES), where SAR1A-GTP-dependent assembly of SEC16A on the ER membrane forms an organized structure that defines these exit sites. SEC16A is essential for normal transitional endoplasmic reticulum (tER) organization and plays roles in both conventional and unconventional protein secretion pathways .

What applications can SEC16A antibodies be used for?

SEC16A antibodies have been validated for multiple research applications including:

These applications allow researchers to detect, quantify, and visualize SEC16A in various experimental contexts .

What is the expected molecular weight of SEC16A in Western blots?

While the calculated molecular weight of SEC16A is 234-252 kDa based on amino acid sequence, it typically appears at 250-300 kDa on Western blots. This size discrepancy may result from post-translational modifications or the protein's structural characteristics affecting electrophoretic mobility .

What is the spatial relationship between SEC16A and other ERES proteins?

High-resolution microscopy studies have revealed that SEC16A occupies spatially distinct but closely juxtaposed positions relative to other COPII components like Sec24C and Sec31A. Immunoelectron microscopy shows that SEC16A and Sec31A typically localize between 100-600 nm apart. While Sec24C and Sec31A show almost complete colocalization, SEC16A forms discrete clusters that are clearly offset from these components in more than 80% of ERES. This spatial arrangement suggests SEC16A may function as an initial organizing platform for ERES formation, with other COPII components assembling in close proximity but not directly overlapping with SEC16A .

How does ER stress affect SEC16A localization and function?

Under ER stress conditions, SEC16A undergoes significant relocalization to the cell periphery, specifically to peripheral ER areas. Quantitative colocalization analysis using Manders' Colocalization Coefficient (MCC) demonstrates increased coincidence between SEC16A and ER marker proteins during stress. This relocalization coincides with SEC16A's association with GRASP55 and facilitates unconventional secretion of proteins like CFTR. The stress response pathway appears to be regulated by IRE1-mediated signaling, which functions upstream of SEC16A during ER stress-induced unconventional secretion .

What are the optimal fixation and antigen retrieval methods for SEC16A immunohistochemistry?

For optimal SEC16A detection in immunohistochemistry applications, tissue sections should undergo antigen retrieval preferably with TE buffer at pH 9.0. Alternatively, citrate buffer at pH 6.0 may be used, though potentially with reduced antigen accessibility. Mouse pancreas tissue has been specifically validated for positive IHC detection of SEC16A. The recommended antibody dilution range for IHC applications is 1:50-1:500 for antibody 29417-1-AP and 1:200-1:1000 for antibody 20025-1-AP, with working concentrations requiring optimization for each experimental system .

How should researchers design co-localization experiments with SEC16A?

When designing co-localization studies for SEC16A with other ERES or trafficking proteins, researchers should:

• Image at the highest spatial resolution possible using confocal microscopy that satisfies Nyquist criteria

• Ensure no pixels are saturated during image acquisition

• Include appropriate markers such as:

  • COPII components (Sec24C, Sec31A) for ERES localization

  • ERGIC-53 for ER-Golgi intermediate compartment

  • COPI components (like β'-COP) for Golgi and retrograde transport

Quantification of co-localization should employ both overlap coefficients and correlation analyses (such as Manders' Colocalization Coefficient) to accurately assess spatial relationships. For highest resolution studies, immunoelectron microscopy using ultrathin cryosections provides definitive spatial mapping at the nanometer scale .

What controls should be included when using SEC16A antibodies for knockdown validation?

When validating SEC16A knockdown experiments, researchers should include:

• Non-targeting siRNA/shRNA controls processed identically to experimental samples

• Positive controls using established SEC16A-expressing cell lines (validated positive WB detection has been reported in HEK-293, HeLa, and HepG2 cells)

• Rescue experiments expressing siRNA-resistant SEC16A constructs to confirm phenotype specificity

• Parallel assessment of multiple SEC16A-dependent processes (conventional and unconventional secretion) to comprehensively evaluate knockdown effects

• Quantification of knockdown efficiency by both protein level (Western blot) and mRNA level (qPCR) analyses

How can researchers address high background in SEC16A immunofluorescence?

High background in SEC16A immunofluorescence experiments may result from several factors. To optimize signal-to-noise ratio:

• Titrate antibody concentration within the recommended range (1:50-1:500 for 20025-1-AP or 1:200-1:800 for 29417-1-AP)

• Extend blocking time using 5% normal serum from the species of the secondary antibody

• Include 0.1-0.3% Triton X-100 during antibody incubation for improved accessibility of the epitope

• Optimize fixation protocols (4% paraformaldehyde for 15 minutes typically works well for SEC16A)

• Increase washing steps and duration between antibody incubations

• Consider using cell lines with validated positive results (A431 cells for 20025-1-AP, HeLa cells for 29417-1-AP)

Why might Western blot detection of SEC16A show multiple bands?

SEC16A detection in Western blots may reveal multiple bands due to:

• Post-translational modifications affecting electrophoretic mobility

• Alternative splicing variants of SEC16A

• Proteolytic degradation during sample preparation

To address this issue, researchers should:

• Use fresh samples with complete protease inhibitor cocktails

• Include phosphatase inhibitors if phosphorylation status is relevant

• Optimize sample denaturing conditions (consider extended boiling times for large proteins like SEC16A)

• Run lower percentage gels (6-8%) to achieve better separation in the high molecular weight range

• Validate specificity using knockout/knockdown controls

• Consider that the observed molecular weight range (250-300 kDa) is higher than the calculated weight (234-252 kDa)

How can researchers improve SEC16A immunoprecipitation efficiency?

To maximize SEC16A immunoprecipitation efficiency:

• Increase starting material (recommended 1.0-3.0 mg of total protein lysate)

• Optimize lysis buffer composition (consider using buffers containing 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris pH 7.5, and 1 mM EDTA)

• Extend antibody incubation time to overnight at 4°C with gentle rotation

• Pre-clear lysates with protein A/G beads prior to adding SEC16A antibody

• Use 0.5-4.0 μg of antibody per immunoprecipitation reaction

• Validate results in HeLa cells, which have been confirmed for positive IP detection

• Consider using crosslinking approaches if studying SEC16A protein complexes

How should researchers interpret SEC16A relocalization during cellular stress?

When analyzing SEC16A relocalization during cellular stress, researchers should consider:

• Quantitative assessment of spatial distribution changes using proper colocalization metrics (e.g., Manders' Colocalization Coefficient)

• Temporal dynamics of relocalization in relation to stress induction

• Correlation with markers of unconventional secretion activation (like GRASP55 association)

• Relationship to IRE1-mediated signaling pathways, which function upstream of SEC16A during ER stress

• Functional consequences on cargo transport through both conventional and unconventional pathways

The peripheral redistribution of SEC16A during stress conditions, particularly its increased association with peripheral ER areas, represents a mechanistic switch from conventional to unconventional secretion pathways rather than simple protein mislocalization .

What approaches can be used to study the functional relationship between SEC16A and GRASP proteins?

To investigate SEC16A-GRASP functional relationships:

• Perform reciprocal co-immunoprecipitation experiments to confirm physical association

• Use proximity ligation assays (PLA) to verify interactions in situ

• Design RNAi experiments targeting each protein individually and in combination

• Employ live-cell imaging with fluorescently tagged proteins to track dynamic interactions

• Utilize super-resolution microscopy techniques to define nanoscale spatial relationships

• Analyze cargo trafficking (e.g., CFTR) as a functional readout of SEC16A-GRASP cooperation

• Assess the effects of ER stress inducers on complex formation and localization

These approaches can help elucidate how SEC16A cooperates with GRASP proteins to facilitate unconventional secretion during cellular stress conditions .

How does SEC16A function differ between cell types and tissues?

While SEC16A is widely expressed, its function and regulation may vary between different cell types and tissues. When interpreting SEC16A research across different biological systems:

• Consider the relative dependence on conventional versus unconventional secretion pathways specific to each cell type

• Examine SEC16A expression levels and splicing variants across tissues (particularly relevant for secretory tissues)

• Assess cell-type specific post-translational modifications that may regulate SEC16A function

• Evaluate interaction partners that may differ between tissues

• Note that positive IHC detection has been specifically validated in mouse pancreas tissue, suggesting particularly relevant functions in this highly secretory organ

• Consider the specialized secretory needs of different cell types that might influence SEC16A dependency

These considerations are essential when extrapolating findings between experimental systems or when developing tissue-specific targeting strategies .