SMC1A Monoclonal Antibody

What is the biological function of SMC1A protein?

SMC1A serves as a central component of the cohesin complex, which is essential for sister chromatid cohesion after DNA replication. The cohesin complex forms a large proteinaceous ring structure that can trap sister chromatids. At anaphase, this complex is cleaved and dissociates from chromatin, allowing proper chromosome segregation . Beyond its structural role in chromosome cohesion, SMC1A is involved in DNA repair through its interaction with BRCA1 and subsequent phosphorylation by ATM, or via phosphorylation by ATR. It functions as a downstream effector in both the ATM/NBS1 and ATR/MSH2 branches of the S-phase checkpoint .

What are the key characteristics of SMC1A monoclonal antibodies?

SMC1A monoclonal antibodies are highly specific immunoglobulins that recognize distinct epitopes on the SMC1A protein. Commercial antibodies typically recognize human SMC1A and may cross-react with mouse and rat homologs . These antibodies are often developed using recombinant protein fragments (such as amino acids 889-1016 of human SMC1A) as immunogens . They are typically supplied in PBS (pH 7.3) containing 1% BSA, 50% glycerol, and 0.02% sodium azide . These antibodies demonstrate high specificity for endogenous levels of SMC1A and do not cross-react with related proteins .

What is the genomic location of SMC1A and how does its expression pattern affect antibody selection?

SMC1A is encoded by an X-linked gene that escapes X-inactivation in humans, resulting in biallelic expression in females . This escape from X-inactivation means that females typically express approximately twice as much SMC1A mRNA as males . When selecting antibodies for comparative studies between sexes, researchers should account for this differential expression pattern, particularly when quantifying protein levels. Additionally, this characteristic makes SMC1A an interesting target for studying sex-specific differences in cellular processes involving chromosome cohesion and DNA repair mechanisms.

How should SMC1A monoclonal antibodies be used for Western blot analysis?

For Western blot applications, SMC1A monoclonal antibodies are typically used at a dilution of 1:500 . The optimal procedure involves:

• Sample preparation: Extract proteins from cells using standard lysis buffers containing protease inhibitors.

• Protein separation: Resolve proteins on an SDS-PAGE gel (typically 7-10% due to SMC1A's large molecular weight of ~143 kDa).

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane.

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

• Primary antibody incubation: Dilute SMC1A antibody 1:500 in blocking buffer and incubate overnight at 4°C.

• Washing: Wash 3-4 times with TBST.

• Secondary antibody incubation: Use an appropriate HRP-conjugated secondary antibody.

• Detection: Visualize using chemiluminescence reagents.

This methodology has been validated for detecting endogenous levels of SMC1A protein in various cell types .

What protocols should be followed for immunohistochemistry with SMC1A antibodies?

For immunohistochemical analysis using SMC1A monoclonal antibodies, the following protocol is recommended based on validated procedures:

• Tissue preparation: Use formalin-fixed, paraffin-embedded tissue sections (4-6 μm thickness).

• Deparaffinization: Remove paraffin and rehydrate sections through graded alcohols.

• Antigen retrieval: Perform heat-induced epitope retrieval, which is critical for optimal staining.

• Blocking: Block endogenous peroxidase activity and non-specific binding.

• Primary antibody: Apply SMC1A monoclonal antibody at a 1:500 dilution and incubate as specified in the antibody datasheet (typically overnight at 4°C).

• Secondary antibody: Apply appropriate detection system.

• Visualization: Develop using DAB (3,3'-diaminobenzidine) and counterstain with hematoxylin.

This approach has been successfully employed for staining SMC1A in various human tissues, including liver tissue and tonsil samples .

How can I analyze SMC1A acetylation status in experimental studies?

To examine SMC1A acetylation levels, researchers can employ immunoprecipitation followed by Western blot analysis:

• Cell lysis: Prepare cell lysates under conditions that preserve protein modifications.

• Immunoprecipitation: Use SMC1A-specific antibodies (such as ab1276 or A300-050A) to pull down SMC1A protein complexes .

• Western blot: Probe the immunoprecipitated material with a pan-specific anti-acetylated lysine antibody (such as CST #9441) .

• Controls: Include appropriate controls to validate specificity (e.g., IgG control, acetylation-deficient mutants).

For more detailed analysis of specific acetylation sites such as K579, which has been implicated in mitotic regulation, site-specific antibodies or mass spectrometry approaches may be necessary .

How can I resolve non-specific binding issues with SMC1A antibodies in Western blots?

Non-specific binding can complicate the interpretation of Western blot results. To address this issue:

• Optimize blocking conditions: Test different blocking agents (BSA vs. milk) and concentrations (3-5%).

• Adjust antibody dilution: Try a more dilute antibody concentration (1:750 or 1:1000) if background is high.

• Increase washing duration and frequency: Perform 4-5 washes with TBST for 10 minutes each.

• Use detergents: Add 0.1-0.3% Triton X-100 to washing buffers to reduce hydrophobic interactions.

• Pre-adsorb the antibody: Incubate with a membrane containing irrelevant proteins before use.

• Validate with positive and negative controls: Include SMC1A-expressing and SMC1A-depleted samples.

If problems persist, consider using alternative SMC1A antibody clones that recognize different epitopes to confirm specificity.

What are the optimal storage conditions for maintaining SMC1A antibody activity?

To maintain optimal antibody activity:

• Short-term storage: Store at 4°C for up to two weeks .

• Long-term storage: Store at -20°C in small aliquots to avoid repeated freeze-thaw cycles .

• Avoid freeze-thaw cycles: Multiple freeze-thaw cycles can significantly reduce antibody activity and specificity.

• Storage buffer: Confirm the antibody is stored in an appropriate buffer (typically PBS with 1% BSA, 50% glycerol, and 0.02% sodium azide) .

• Handling: When using frozen aliquots, thaw on ice and centrifuge briefly before use.

Following these guidelines will help preserve antibody integrity and ensure consistent experimental results over time.

How can SMC1A antibodies be used to investigate the phosphorylation-dependent functions of SMC1A?

SMC1A undergoes ATM/ATR-dependent phosphorylation at several key residues, including Ser957 and Ser966, in response to DNA damage . To investigate these phosphorylation events:

• Phospho-specific antibodies: Use phospho-specific antibodies that recognize Ser957 or Ser966 phosphorylation sites (e.g., Rockland 200-301-397) .

• Sequential immunoprecipitation: First immunoprecipitate total SMC1A, then probe with phospho-specific antibodies.

• Lambda phosphatase treatment: Compare antibody reactivity before and after phosphatase treatment to confirm specificity.

• Mutational analysis: Compare wild-type SMC1A with phospho-deficient mutants (S957A, S966A).

• DNA damage induction: Monitor phosphorylation kinetics following treatment with DNA-damaging agents.

This approach can provide insights into how phosphorylation regulates SMC1A function in DNA damage response pathways.

What are the considerations for investigating SMC1A in the context of cohesin complex dynamics?

When studying SMC1A as part of the cohesin complex:

• Co-immunoprecipitation: Use SMC1A antibodies to pull down the entire cohesin complex, followed by probing for other components (SMC3, RAD21, STAG1/2).

• Chromatin immunoprecipitation (ChIP): Employ SMC1A antibodies for ChIP assays to identify genomic binding sites of the cohesin complex.

• Proximity ligation assays: Visualize protein-protein interactions between SMC1A and other cohesin components in situ.

• Cell cycle synchronization: Analyze cohesin complex composition and SMC1A modifications at different cell cycle stages.

• Depletion strategies: Compare partial vs. complete SMC1A depletion to distinguish between structural and regulatory roles.

These approaches can help elucidate the dynamic associations between SMC1A and other proteins throughout the cell cycle and in response to various cellular stresses.

How can researchers investigate the role of SMC1A acetylation in regulating mitotic progression?

The acetylation status of SMC1A, particularly at lysine 579, has been implicated in mitotic regulation through the SIRT2-SMC1A axis . To investigate this relationship:

• Acetylation-mimetic mutants: Compare wild-type SMC1A with acetylation-mimetic (K579Q) mutants in functional assays.

• Deacetylase inhibitors: Treat cells with SIRT2-specific inhibitors to promote SMC1A acetylation.

• Microscopy: Analyze spindle formation and chromosome segregation using immunofluorescence microscopy.

• Protein-protein interaction studies: Assess how acetylation affects SMC1A interactions with mitotic regulators like Rae1 .

• Cell cycle analysis: Monitor mitotic progression in cells expressing wild-type versus acetylation-mimetic SMC1A variants.

Research has shown that hyperacetylation of SMC1A at K579 leads to mitotic arrest with defective mitotic features and ultimately cell apoptosis , making this an important area for cancer research.

What approaches can be used to resolve contradicting data regarding SMC1A expression and mutation effects?

When facing contradictory data regarding SMC1A:

• Sex-specific analysis: Given that SMC1A escapes X-inactivation, separate data analysis by sex, as females express approximately twice as much SMC1A mRNA as males .

• Allele-specific expression: For heterozygous mutations, employ allele-specific quantitative RT-PCR to distinguish between wild-type and mutant allele expression levels .

• Tissue-specific effects: Compare SMC1A expression and function across different cell types and tissues.

• Mutation-specific effects: Different mutations may have distinct functional consequences despite affecting the same protein.

• Technical validation: Validate findings using multiple antibodies targeting different epitopes and alternative detection methods.

Studies have shown that expression levels of SMC1A can vary significantly between individuals and families, which may contribute to contradictory results in the literature .

How can SMC1A antibodies be utilized in studying Cornelia de Lange syndrome (CdLS)?

Mutations in SMC1A are associated with Cornelia de Lange syndrome, a developmental disorder characterized by distinctive facial features, growth delays, and intellectual disability . Researchers investigating CdLS can utilize SMC1A antibodies to:

• Compare protein levels and localization in patient-derived cells versus controls.

• Assess the impact of specific mutations on protein-protein interactions and complex formation.

• Evaluate post-translational modifications in patient samples compared to controls.

• Perform ChIP-seq to identify differential chromatin binding patterns of mutant SMC1A proteins.

• Develop mutation-specific antibodies for diagnostic purposes.

Studies have shown that CdLS-associated SMC1A mutations are typically missense or small in-frame deletions that preserve the open reading frame, suggesting a dominant-negative mechanism rather than haploinsufficiency .

This table summarizes key differences observed between CdLS patients with SMC1A mutations versus those with NIPBL mutations, based on clinical studies .