SEC24A Antibody

What is SEC24A and why are antibodies against it important in research?

SEC24A (SEC24 homolog A, COPII coat complex component) is a protein encoded by the SEC24A gene in humans. It functions as an adaptor protein for secretory proteins during COPII-mediated vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus . The protein is approximately 119.7 kilodaltons in mass and plays a critical role in cargo selection during vesicle formation .

Antibodies against SEC24A are essential research tools that allow scientists to detect, quantify, and visualize this protein in various experimental contexts. These antibodies have revealed SEC24A's involvement in multiple cellular processes, including recently identified roles in thapsigargin-induced cell death pathways .

What are the common applications for SEC24A antibodies in laboratory research?

SEC24A antibodies are utilized across multiple experimental applications:

These applications allow researchers to investigate SEC24A's expression, localization, and interactions within cellular contexts .

What species reactivity should I consider when selecting a SEC24A antibody?

When selecting a SEC24A antibody, species reactivity is a crucial consideration to ensure compatibility with your experimental model. Available SEC24A antibodies demonstrate reactivity across several species:

• Human (Hu): Most widely available and validated

• Monkey (Mk): Often cross-reactive with human antibodies

• Mouse (Ms): Available for murine research models

• Rat (Rt): Available for rat-based experimental systems

• Porcine (Pr): Less common but available from select suppliers

Always verify the specific epitope and validate cross-reactivity when working with model organisms, as sequence conservation varies across regions of the protein .

How can I distinguish between SEC24A and other SEC24 family members in my experiments?

Distinguishing between SEC24 family members (SEC24A, B, C, and D) requires careful antibody selection and experimental design:

• Epitope selection: Choose antibodies raised against unique regions of SEC24A. N-terminal targeted antibodies often provide better specificity, as this region shows greater sequence divergence between family members .

• Validation methods:

  • Perform siRNA knockdown of SEC24A followed by Western blot to confirm specificity

  • Use SEC24A-/- cell lines as negative controls

  • Compare staining patterns with known SEC24A subcellular distribution

• Cross-reactivity testing: Some antibodies, like the one from Cell Signaling Technology (#9678), are specifically engineered not to cross-react with other SEC24 family members, making them ideal for selective detection .

• Combined detection approaches: Use orthogonal methods to confirm specificity, such as mass spectrometry validation following immunoprecipitation.

What is the role of SEC24A in thapsigargin-induced cell death, and how can antibodies help elucidate this mechanism?

Recent research has identified SEC24A as an essential mediator of thapsigargin-induced cell death in HAP1 cells through a genome-wide CRISPR/Cas9 screen . This represents a novel function beyond its canonical role in COPII vesicle transport.

Antibodies can help elucidate this mechanism through:

• Co-immunoprecipitation studies: Using SEC24A antibodies to pull down protein complexes during thapsigargin treatment can reveal novel interaction partners specific to this death pathway.

• Time-course immunofluorescence: Tracking SEC24A localization changes before and after thapsigargin treatment using IF can reveal dynamic subcellular movements relevant to cell death induction.

• Phosphorylation-specific antibodies: Developing or using phospho-specific SEC24A antibodies could identify post-translational modifications associated with its role in cell death signaling.

• Proximity ligation assays: Combining SEC24A antibodies with antibodies against ER stress mediators to identify specific protein-protein interactions during stress response .

These approaches have revealed that SEC24A likely acts upstream of the unfolded protein response (UPR) to mediate cell death from SERCA inhibition, though the exact mechanism remains under investigation .

How do mutations or altered expression of SEC24A affect its detection by antibodies in disease models?

Mutations or altered expression of SEC24A in disease models can significantly impact antibody recognition, leading to potential experimental artifacts:

• Epitope masking: Post-translational modifications (phosphorylation, ubiquitination) at or near antibody binding sites can block antibody access. This is particularly relevant in stress conditions when SEC24A may undergo regulatory modifications.

• Conformational changes: Disease-associated mutations may alter protein folding, potentially exposing or concealing epitopes recognized by certain antibodies.

• Expression level variations: In disease models where SEC24A is significantly upregulated or downregulated, antibody titration may be necessary to maintain detection within the linear range.

• Truncated variants: Some disease states may produce truncated forms of SEC24A, requiring antibodies targeting different protein regions for comprehensive detection.

To address these challenges:

• Use multiple antibodies targeting different epitopes

• Include appropriate positive and negative controls from both healthy and disease tissues

• Validate findings with complementary techniques such as mass spectrometry or mRNA expression analysis

What are the optimal conditions for western blotting with SEC24A antibodies?

Optimizing western blot conditions for SEC24A detection requires attention to several key parameters:

Sample preparation:

• Use RIPA or NP-40 buffer with protease inhibitors

• Include phosphatase inhibitors if studying phosphorylation states

• Optimal protein load: 20-50 μg of total protein from cell lysates

Gel electrophoresis and transfer:

• Use 8% or 10% SDS-PAGE gels due to SEC24A's large size (120 kDa)

• Transfer at lower voltage (30V) overnight at 4°C to ensure complete transfer of large proteins

Antibody conditions:

• Primary antibody dilution: Typically 1:1000 as recommended for Cell Signaling Technology's SEC24A antibody

• Incubation: Overnight at 4°C with gentle rocking

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

Detection considerations:

• SEC24A appears as a prominent band at approximately 120 kDa

• Secondary antibody selection should match the host species of your primary antibody

• For low expression samples, consider enhanced chemiluminescence substrates

What controls should be included when using SEC24A antibodies for immunoprecipitation studies?

When performing immunoprecipitation with SEC24A antibodies, include these essential controls:

• Input control: 5-10% of pre-immunoprecipitation lysate to confirm target protein presence

• Isotype control: Immunoprecipitation with non-specific IgG from the same species as the SEC24A antibody to identify non-specific binding

• Negative cell line control: Utilize cells with CRISPR/Cas9 knockout or siRNA knockdown of SEC24A

• Pre-clearing step: Pre-clear lysates with protein A/G beads alone to reduce non-specific binding

• Validation controls: If studying interactions, perform reverse immunoprecipitation with antibodies against suspected interacting partners

The Cell Signaling Technology SEC24A antibody (#9678) has been specifically validated for immunoprecipitation applications with a recommended dilution of 1:50 .

How can SEC24A antibodies be used to study the protein's role in COPII vesicle transport?

SEC24A antibodies provide valuable tools for investigating COPII vesicle transport through multiple experimental approaches:

• Co-localization studies:

  • Perform dual immunofluorescence with SEC24A antibodies and markers for ER exit sites (SEC16), ERGIC (ERGIC-53), or Golgi (GM130)

  • Quantify co-localization coefficients under normal and stress conditions

• Live-cell imaging:

  • Generate GFP-SEC24A fusion constructs and validate expression using SEC24A antibodies

  • Track vesicle formation and movement in real-time, correlating with fixed-cell antibody staining

• Immunoelectron microscopy:

  • Use gold-conjugated secondary antibodies against SEC24A primaries to visualize SEC24A at the ultrastructural level on forming COPII vesicles

• Cargo selection analysis:

  • Immunoprecipitate SEC24A under varying conditions to identify bound cargo proteins

  • Compare changes in cargo binding efficiency when specific residues are mutated

• Pulse-chase experiments:

  • Track protein transport from ER to Golgi using synchronized cargo release

  • Use SEC24A antibodies to correlate vesicle formation with cargo movement

These approaches have revealed that SEC24A has specialized roles in the transport of specific cargo proteins distinct from other SEC24 family members, contributing to our understanding of selective protein export from the ER .

What troubleshooting approaches should be considered when SEC24A antibodies show unexpected results?

When SEC24A antibodies produce unexpected results, consider these systematic troubleshooting strategies:

• Verification of antibody specificity:

  • Perform peptide competition assays with the immunizing peptide

  • Test on known positive and negative control samples

  • Verify results with a second antibody targeting a different epitope

• Sample preparation issues:

  • For membrane-associated fractions of SEC24A, ensure adequate detergent solubilization

  • Check protein degradation by including protease inhibitors and maintaining cold temperatures

  • Consider native vs. denaturing conditions for maintaining epitope integrity

• Technical parameters:

  • Titrate antibody concentration beyond recommended dilutions

  • Adjust incubation time and temperature

  • For IHC/IF, test multiple fixation methods (paraformaldehyde vs. methanol)

• Biological variables:

  • Consider cell cycle dependence of SEC24A expression

  • Evaluate stress conditions that might alter SEC24A localization or complex formation

  • Check for tissue-specific isoforms that might not be recognized by all antibodies

• Post-translational modifications:

  • If unexpected band sizes appear, investigate potential ubiquitination or SUMOylation

  • Consider phosphorylation status, particularly after cellular stress

How have SEC24A antibodies contributed to understanding the protein's role beyond vesicular transport?

Recent research utilizing SEC24A antibodies has revealed several non-canonical functions:

• Cell death pathways: SEC24A has been identified as a critical mediator of thapsigargin-induced cell death, acting upstream of the unfolded protein response. Antibodies have helped track its redistribution during ER stress .

• Calcium signaling: SEC24A appears to facilitate colocalization and calcium flux between cellular compartments. Immunofluorescence studies with SEC24A antibodies have mapped these interactions spatially .

• Developmental processes: Studies in model organisms have implicated SEC24A in tissue-specific developmental pathways. Antibodies have helped define expression patterns across tissues and developmental stages .

• Disease associations: Altered SEC24A expression has been linked to several pathological conditions, with antibodies providing crucial tools for comparative expression studies.

These discoveries highlight SEC24A's multifunctional nature beyond its canonical role in COPII vesicle transport, opening new research directions for understanding cellular homeostasis.

What emerging techniques are enhancing the utility of SEC24A antibodies in advanced research applications?

Emerging techniques are expanding the research applications of SEC24A antibodies:

• Proximity labeling approaches:

  • BioID or APEX2 fusions with SEC24A allow identification of proximal proteins in living cells

  • Results can be validated using conventional SEC24A antibodies in co-immunoprecipitation studies

• Super-resolution microscopy:

  • Techniques like STORM and STED provide nanoscale visualization of SEC24A at ER exit sites

  • Dual-color super-resolution with SEC24A antibodies and cargo markers reveals spatial organization of early secretory pathway

• Single-cell proteomics:

  • SEC24A antibodies coupled with mass cytometry (CyTOF) enable analysis of expression heterogeneity

  • Correlation with other secretory pathway markers at single-cell resolution

• Intrabodies and nanobodies:

  • Development of intracellularly expressed antibody fragments against SEC24A enables live-cell tracking

  • Smaller binding fragments provide improved spatial resolution and reduced steric hindrance

• Antibody-guided CRISPR screening:

  • SEC24A antibodies used to sort cells based on expression levels before or after genome-wide screens

  • Enables identification of genes affecting SEC24A stability, localization, or function

These advanced applications are providing unprecedented insights into SEC24A biology and its roles in cellular homeostasis.