SEC31A Antibody

What is SEC31A and why is it important in cellular biology?

SEC31A (also known as ABP125, ABP130, SEC31L1) is a 1,220 amino acid protein (approximately 133 kDa) characterized by seven WD repeats that typically form a tertiary propeller configuration . It functions as a critical component of the COPII (coat protein II) complex, which is responsible for vesicle budding from the endoplasmic reticulum (ER) .

SEC31A plays two essential roles in vesicular transport:

• Physical deformation of the endoplasmic reticulum membrane into vesicles

• Selection of cargo molecules for transport from the ER to the Golgi apparatus

In mammalian cells, SEC31A interacts with SEC13 to form the outer coat of COPII vesicles, which is crucial for maintaining cellular homeostasis through proper protein trafficking . This process represents a fundamental cellular mechanism for protein transport.

Proper storage and handling are critical for maintaining antibody functionality:

• Storage temperature: Store at -20°C for most formulations

• Buffer composition: Most SEC31A antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Stability: Generally stable for one year after shipment when stored properly

• Aliquoting: While some manufacturers note that aliquoting is unnecessary for -20°C storage, dividing into single-use aliquots is recommended to prevent freeze-thaw cycles

• Special considerations for fluorescent conjugates: For conjugated antibodies like CL488-17913, avoid exposure to light and note that storage buffer may contain additional components (0.05% Proclin300, 0.5% BSA)

What are the best practices for validating SEC31A antibody specificity?

Validation of antibody specificity is essential for reliable research outcomes. The following methodologies have proven effective for SEC31A antibody validation:

• Knockdown/Knockout Verification:

  • Several publications have utilized SEC31A siRNA (targeting nucleotide positions 872-890 or 907-925) to demonstrate antibody specificity

  • Construct vectors targeting these regions using systems like pSilencer 1.0-U6 (Ambion)

  • Transfect cells twice at 48-hour intervals for optimal knockdown efficiency

• Western Blot Analysis:

  • Expected molecular weight: 118-134 kDa (observed range varies slightly between antibodies)

  • Use HeLa cell lysate at concentrations of 5-50 μg for optimal detection

  • Some antibodies like ab86600 have been validated at concentrations as low as 0.04 μg/mL

• Immunofluorescence Pattern Recognition:

  • Authentic SEC31A staining should show vesicular structures in the perinuclear region

  • Co-localization with other COPII components like SEC13 provides additional validation

How can I optimize SEC31A antibody performance in immunofluorescence applications?

For optimal immunofluorescence results with SEC31A antibodies:

• Sample Preparation Protocol:

  • Fix cells using PFA (paraformaldehyde)

  • Permeabilize with Triton X-100 for optimal antigen accessibility

  • U-251MG (Human brain glioma) and HeLa cells have been successfully used as positive controls

• Dilution Optimization:

  • Begin with manufacturer's recommended range (typically 1:200-1:800)

  • For conjugated antibodies like CL488-17913, a wider range (1:50-1:500) may be tested

  • Titrate to determine optimal signal-to-noise ratio for your specific experimental system

• Expected Localization Pattern:

  • SEC31A localizes to COPII-positive vesicular structures concentrated in the perinuclear region

  • This pattern is altered after calcium chelation, which can serve as a specificity control

What is known about SEC31A's role in disease processes and how can antibodies help investigate these mechanisms?

Recent research has revealed important connections between SEC31A and disease:

• Cancer Biology Applications:

  • CircSEC31A (circular RNA derived from SEC31A) promotes progression in non-small cell lung cancer (NSCLC)

  • Knockdown of circSEC31A has been shown to suppress:

    • NSCLC cell migration and invasion

    • Glycolysis

    • Tumor growth in vivo

  • Research indicates a positive correlation between SEC31A expression levels and circSEC31A in NSCLC tissues

• Experimental Approaches:

  • Combined qRT-PCR and Western blot analysis using SEC31A antibodies can assess both mRNA and protein expression levels

  • Subcellular fractionation assays help determine the localization of SEC31A and its derivatives

  • Transwell assays can measure changes in cell migration and invasion following SEC31A manipulation

• Clinical Correlations:

  • CircSEC31A expression has been associated with:

    • Tumor size

    • TNM stage

    • Lymphatic metastasis

  • Chromosomal aberrations involving SEC31A have been linked to inflammatory myofibroblastic tumors

How does calcium signaling regulate SEC31A function and how can this be studied?

The calcium-binding protein ALG-2 interacts with SEC31A in a calcium-dependent manner, representing an important regulatory mechanism:

• Interaction Mechanism:

  • ALG-2 binds to the Pro-rich region of SEC31A specifically in a Ca²⁺-dependent manner

  • This interaction affects the retention kinetics of SEC31A at ER exit sites

  • Anti-ALG-2 antibodies can co-precipitate SEC31A from cell lysates even without exogenously added Ca²⁺

• Experimental Approaches:

  • Live-cell time-lapse imaging using GFP-fused ALG-2 and RFP-fused SEC31A constructs can visualize this interaction dynamics

  • Treatment with cell-permeable Ca²⁺ chelators causes mislocalization of ALG-2 and reduced SEC31A at ER exit sites

  • Ca²⁺-binding deficient ALG-2 mutants, which do not bind SEC31A, serve as valuable negative controls

• Functional Consequences:

  • ALG-2 depletion substantially reduces the level of SEC31A associated with the membrane at ER exit sites

  • This suggests calcium signaling may regulate COPII vesicle formation through modulation of SEC31A recruitment

What are the critical technical considerations when performing Western blot analysis with SEC31A antibodies?

For optimal Western blot results with SEC31A antibodies:

Additional considerations:

• Use appropriate positive controls (HeLa cells are most frequently validated)

• Some SEC31A antibodies are available as direct HRP conjugates, which may simplify protocols

• For co-immunoprecipitation studies, protein A/G beads with SEC31A antibodies have shown effectiveness

How do mutations or altered expression of SEC31A impact cellular function?

Understanding the impact of SEC31A disruption is valuable for both basic research and disease-related studies:

• Knockdown Effects:

  • SEC31A depletion via siRNA impairs ER-to-Golgi transport

  • Experiments demonstrate that immunodepletion of SEC31A specifically blocks vesicular transport

  • Effects can be observed through disruption of vesicular stomatitis G protein transport, a standard assay for ER-Golgi trafficking

• Structural Requirements:

  • The WD-40 domain in the N-terminal region is critical for function

  • The Pro-rich C-terminal region mediates protein-protein interactions, particularly with regulatory factors like ALG-2

• Tissue-Specific Considerations:

  • SEC31A transcripts are ubiquitously expressed, while the homolog SEC31B shows tissue-specific expression (primarily in testis and thymus)

  • This suggests potential tissue-specific roles that may require careful experimental design when studying different cell types

What are common issues when using SEC31A antibodies and how can they be addressed?

For application-specific recommendations:

• Western blot: Follow standard protocols as referenced at BD Biosciences website

• Immunofluorescence: Use dilutions of 1:50-1:500 for conjugated antibodies, 1:200-1:800 for unconjugated

• Immunoprecipitation: Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

What alternative approaches can complement SEC31A antibody-based methods?

For comprehensive analysis of SEC31A, consider these complementary approaches:

• RNA-based methods:

  • qRT-PCR for SEC31A mRNA expression quantification

  • siRNA knockdown targeting positions 872-890 or 907-925

  • Analysis of circSEC31A, which has distinct functions from the linear transcript

• Protein fusion systems:

  • GFP-tagged SEC31A for live cell imaging

  • RFP-fused SEC31A for co-localization studies

  • These approaches have been successfully used to study SEC31A dynamics at ER exit sites

• Functional assays:

  • Measure vesicular transport efficiency using reporter proteins

  • Assess glycolysis parameters (glucose consumption, lactate production, ATP levels) to link SEC31A to metabolic functions

  • Transwell assays to evaluate effects on cell migration and invasion

What emerging research areas involve SEC31A antibodies?

Recent findings suggest several promising research directions where SEC31A antibodies will be valuable tools:

• Role in Cancer Biology:

  • CircSEC31A has been identified as a potential oncogenic factor in NSCLC

  • Further studies may reveal mechanisms connecting vesicular transport defects to cancer progression

  • Investigation of SEC31A as a potential biomarker or therapeutic target

• Calcium Signaling Integration:

  • The calcium-dependent regulation of SEC31A via ALG-2 represents an important area for further study

  • This connection may link calcium signaling pathways to fundamental membrane trafficking processes

• Alternative Splicing Regulation:

  • Multiple isoforms of SEC31A exist due to alternative splicing

  • Understanding the functional differences between these isoforms could reveal regulatory mechanisms

  • SEC31A antibodies that can distinguish between isoforms would be particularly valuable for such research