STAG1 Antibody

What is STAG1 and what cellular functions does it regulate?

STAG1, also known as stromal antigen 1 or cohesin subunit SA-1, is a 144.4 kilodalton protein that functions as a critical subunit of the cohesin complex. This complex plays essential roles in regulating sister chromatid cohesion and DNA looping, while also contributing to spindle pole assembly and chromatid cleavage during the anaphase stage of mitosis . STAG1 is also known by alternative names including MRD47, SA1, SCC3A, and SCC3 homolog 1, which reflects its evolutionary conservation and functional importance . The protein has well-characterized orthologs in multiple model organisms including canine, porcine, monkey, mouse, and rat species, making it suitable for comparative studies across different experimental systems .

What detection methods are compatible with STAG1 antibodies?

STAG1 antibodies have been validated for multiple experimental applications, with Western blot (WB) being the most commonly supported technique across commercial antibodies. Additional applications include immunoprecipitation (IP), immunohistochemistry (IHC), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) . When selecting an appropriate antibody, researchers should consider the specific applications they intend to use. For instance, the GeneTex anti-SA1 antibody is validated for WB and IP applications, while the Bethyl Laboratories rabbit anti-SA1 antibody is suitable for WB, IHC, and IP methods . The Boster Bio STAG1 antibody has been validated for Western blot, immunofluorescence, and flow cytometry applications with specific protocols available for each technique .

How should STAG1 antibody validation be performed for Western blot applications?

Proper validation of STAG1 antibodies for Western blot applications requires several critical steps. First, sample preparation should begin with efficient cell lysis under reducing conditions and loading approximately 30 μg of protein per lane . Electrophoresis should be performed on SDS-PAGE gels with appropriate percentage (e.g., 5-20% gradient gels) at 70-90V for 2-3 hours to achieve optimal separation of this large protein . Following transfer to nitrocellulose membranes (typically at 150 mA for 50-90 minutes), blocking with 5% non-fat milk in TBS for 1.5 hours at room temperature is recommended . Primary antibody incubation should be conducted overnight at 4°C with concentration-optimized antibody (e.g., 0.5 μg/mL), followed by thorough washing and appropriate secondary antibody incubation . For detection, enhanced chemiluminescence systems should be employed, with expected band size for STAG1 at approximately 144-155 kDa .

What are the optimal conditions for STAG1 immunofluorescence staining in different cell types?

For immunofluorescence detection of STAG1, enzyme antigen retrieval methods have shown superior results compared to heat-based methods. According to validated protocols, enzyme antigen retrieval should be performed for approximately 15 minutes prior to blocking with 10% goat serum . Primary antibody concentration should be optimized, with 5 μg/mL being effective in established protocols . For dual staining applications, STAG1 antibodies can be effectively paired with other cellular markers such as anti-Tubulin Alpha antibodies to provide contextual cellular localization information . Secondary antibody selection should be guided by the host species of the primary antibody, with fluorophore-conjugated antibodies (e.g., Cy3-conjugated anti-rabbit IgG) being suitable for STAG1 visualization. For optimal results, secondary antibody incubation should be performed at 37°C for 30 minutes at dilutions of approximately 1:500 . Nuclear counterstaining can be performed to provide context for STAG1's nuclear localization pattern, which reflects its role in chromatin organization.

How can STAG1 antibodies be effectively used in flow cytometry applications?

For flow cytometry applications using STAG1 antibodies, cell fixation and permeabilization are critical preprocessing steps due to STAG1's predominantly intracellular localization. A validated protocol involves fixing cells with 4% paraformaldehyde followed by permeabilization with an appropriate buffer system . Cell blocking should be performed with 10% normal goat serum prior to primary antibody incubation . For optimal staining, incubate cells with rabbit anti-STAG1 antibody at a concentration of 1 μg per 1×10^6 cells for 30 minutes at 20°C . For detection, use fluorophore-conjugated secondary antibodies such as DyLight®488-conjugated goat anti-rabbit IgG at 5-10 μg per 1×10^6 cells for 30 minutes at 20°C . Appropriate controls must be included, such as isotype control antibodies (e.g., rabbit IgG at equivalent concentration) and unstained samples to establish baseline fluorescence and confirm specificity .

What are the considerations for using STAG1 antibodies in immunoprecipitation experiments?

Immunoprecipitation (IP) experiments with STAG1 antibodies require special consideration due to the protein's involvement in multi-protein complexes and its relatively large size. When performing IP for STAG1, non-denaturing lysis buffers containing mild detergents should be used to preserve protein-protein interactions within the cohesin complex . Antibodies specifically validated for IP applications, such as the GeneTex anti-SA1 antibody or Bethyl Laboratories rabbit anti-SA1 antibody, should be selected . Pre-clearing of lysates with protein A/G beads is recommended to reduce non-specific binding. For optimal results, antibody concentrations must be carefully titrated, with approximately 2-5 μg of antibody per 500 μg of total protein being a reasonable starting point. Extended incubation times (overnight at 4°C) may improve the efficiency of STAG1 immunoprecipitation. When analyzing co-immunoprecipitated proteins, researchers should consider the known interacting partners of STAG1 within the cohesin complex to validate their experimental approach.

How can researchers address weak or non-specific signals in STAG1 Western blots?

When encountering weak signals in STAG1 Western blots, several optimization strategies can be implemented. First, ensure adequate protein loading (30 μg or more per lane) as recommended in validated protocols . For large proteins like STAG1 (144.4 kDa), extended transfer times (50-90 minutes at 150 mA) may improve protein transfer efficiency to the membrane . If signal remains weak, antibody concentration can be increased incrementally, though this should be balanced against potential increases in background signal. Non-specific signals can be addressed by optimizing blocking conditions (increasing blocking time to 1.5 hours or longer) and implementing more stringent washing protocols between antibody incubations . Additionally, confirming the expected molecular weight (approximately 155 kDa for STAG1) is crucial for distinguishing specific from non-specific signals . If high background persists, reducing primary and secondary antibody concentrations, extending washing steps, or using alternative blocking agents may improve signal-to-noise ratio.

What approaches can resolve cross-reactivity issues with STAG1 antibodies across species?

Cross-reactivity considerations are particularly important when using STAG1 antibodies across different species. While human STAG1 antibodies may cross-react with orthologs from other mammals due to sequence conservation, validation for each specific species is essential . When selecting antibodies for cross-species applications, prioritize those with demonstrated reactivity across multiple species, such as the Aviva Systems Biology STAG1 antibody that has been validated for human, mouse, rabbit, rat, bovine, dog, guinea pig, and horse samples . For unvalidated species, preliminary titration experiments should be performed to determine optimal antibody concentrations. Epitope mapping can help identify regions of high conservation across species that might serve as optimal targets for antibody binding. In cases where cross-reactivity is not established, species-specific antibodies should be utilized, particularly for critical experiments where precise target recognition is essential.

How should researchers interpret variations in STAG1 band patterns between different cell lines?

Variations in STAG1 band patterns across different cell lines may reflect biological differences rather than technical artifacts. When comparing STAG1 expression across cell lines, such as HEL and HeLa cells in published validations, consistent detection at the expected molecular weight (approximately 155 kDa) should be observed despite potential intensity variations . Differences in band intensity may reflect varying expression levels of STAG1 across cell types, while the appearance of additional bands might indicate post-translational modifications, alternative splicing variants, or protein degradation. To distinguish between these possibilities, researchers should consider performing additional experiments, such as siRNA knockdown of STAG1 to confirm band specificity, or phosphatase treatment to identify phosphorylated forms of the protein. Cell type-specific variations in STAG1 expression may have biological significance related to the protein's role in chromosome cohesion and DNA repair pathways, making these observations potentially valuable research findings rather than merely technical concerns.

How can STAG1 antibodies be used to investigate chromatin organization and looping?

STAG1 antibodies can be powerful tools for studying chromatin organization due to STAG1's role in the cohesin complex that regulates DNA looping . For chromatin immunoprecipitation (ChIP) applications, researchers should select antibodies specifically validated for this technique. The experimental approach should include crosslinking of protein-DNA complexes with formaldehyde, followed by cell lysis and sonication to generate chromatin fragments of appropriate size (typically 200-500 bp). Immunoprecipitation with STAG1 antibodies will enrich for genomic regions associated with STAG1-containing cohesin complexes. Sequential ChIP (ChIP-reChIP) can be performed to identify genomic regions bound by STAG1 in combination with other cohesin components or chromatin regulators. For genome-wide analyses, ChIP followed by next-generation sequencing (ChIP-seq) can identify the global distribution of STAG1 binding sites across the genome. Data analysis should focus on identifying enrichment patterns at specific genomic features such as enhancers, promoters, and CTCF binding sites, which can provide insights into STAG1's role in three-dimensional genome organization.

What methodological approaches can distinguish between STAG1 and STAG2 functions in experimental systems?

Distinguishing between STAG1 and STAG2 functions requires careful selection of antibodies with confirmed specificity for each paralog. When designing experiments to differentiate their roles, researchers should first validate antibody specificity using knockout or knockdown cell lines for each protein. For functional studies, selective depletion of either STAG1 or STAG2 using siRNA or CRISPR-Cas9 approaches, followed by rescue experiments with the complementary protein, can help identify unique versus redundant functions. Co-immunoprecipitation experiments using STAG1-specific antibodies can identify protein interaction partners that may differ from those of STAG2. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) with paralog-specific antibodies can reveal distinct genomic binding patterns between STAG1 and STAG2. When analyzing data, researchers should focus on identifying both overlapping and distinct phenotypes or genomic binding sites to fully characterize the unique contributions of each protein to cohesin complex function and chromatin organization.

What are the recommended protocols for using STAG1 antibodies in tissue sections versus cultured cells?

The application of STAG1 antibodies in tissue sections requires modifications to protocols optimized for cultured cells. For tissue sections, more rigorous antigen retrieval methods are typically necessary, with enzyme-based approaches showing good results for STAG1 detection . Tissue-specific optimization of antibody concentration is essential, with starting concentrations approximately 1.5-2× higher than those used for cultured cells. Incubation times may need to be extended for tissue sections to ensure adequate antibody penetration. When performing dual immunofluorescence in tissues, careful selection of compatible antibody pairs is critical to minimize cross-reactivity. The following table summarizes the key differences in protocols between cultured cells and tissue sections:

When analyzing results, researchers should consider the cellular heterogeneity of tissues compared to cultured cell lines, which may result in more variable staining patterns requiring careful interpretation.

How can STAG1 antibodies contribute to understanding cohesin complex dysregulation in disease states?

STAG1 antibodies offer valuable tools for investigating cohesin complex dysregulation in various disease states, particularly in cancer and developmental disorders. Researchers can employ STAG1 antibodies in immunohistochemistry of patient-derived samples to assess expression levels and localization patterns that may correlate with disease progression or prognosis. Comparative analysis of STAG1 binding patterns in normal versus disease tissues using ChIP-seq can reveal alterations in chromatin architecture associated with pathological states. Proximity ligation assays utilizing STAG1 antibodies paired with antibodies against other cohesin components can detect abnormal protein-protein interactions within the complex in disease contexts. Researchers should design studies that correlate STAG1 binding or expression patterns with specific disease phenotypes or clinical outcomes. Advanced quantitative approaches, including digital pathology analysis of STAG1 immunostaining or integrated multi-omics analyses incorporating STAG1 ChIP-seq data, can provide comprehensive insights into the role of cohesin dysregulation in disease pathogenesis and potentially identify new therapeutic targets.