ska2 Antibody

What is SKA2 and what are its primary cellular functions?

SKA2 (Spindle and Kinetochore-Associated protein 2), also known as FAM33A, is a crucial 121 amino acid component of the SKA complex that plays an essential role in mitosis. The protein localizes to the outer kinetochore and spindle microtubules during cell division where it performs several critical functions:

• Forms part of the microtubule-binding subcomplex at the kinetochore-microtubule interface

• Facilitates stable kinetochore-microtubule interactions necessary for proper chromosome segregation

• Enables spindle checkpoint silencing and transition out of mitosis

• Interacts with glucocorticoid receptor (GR) to modulate GC signaling

SKA2 is encoded by a gene located on human chromosome 17, a region encompassing over 2.5% of the human genome .

How does SKA2 expression vary across different tissue types?

SKA2 expression exhibits significant variation across different cell lines and tissues:

• Wide variation across multiple cell lines

• Not detected in the liver cell line HepG2

• High expression in small cell lung cancer (SCLC) xenograft models

• Present in several human lung and breast tumors

• Found predominantly in the cytoplasm in most cell types, where it co-localizes with glucocorticoid receptor (GR)

• Nuclear expression observed specifically in breast tumors

What are the basic characteristics of commercially available SKA2 antibodies?

Available SKA2 antibodies include both polyclonal and monoclonal varieties with these general specifications:

Both conjugated (HRP, PE, FITC, Alexa Fluor®) and unconjugated formats are commercially available .

What are the optimal conditions for detecting SKA2 by Western blot?

For optimal Western blot detection of SKA2:

• Sample preparation:

  • Use freshly prepared cell lysates whenever possible

  • Include protease inhibitors during lysis to prevent degradation

  • Maintain cold temperature throughout sample processing

• Electrophoresis and transfer:

  • Use 12-15% gels due to SKA2's relatively small size (14 kDa calculated MW)

  • Note that observed molecular weight may appear higher (up to 68 kDa has been reported)

  • Use PVDF membranes for optimal protein binding

• Antibody incubation:

  • Starting dilution: 0.5-1 μg/ml for primary antibody

  • Extend primary antibody incubation to overnight at 4°C

  • Use 5% non-fat milk or BSA in TBS-T for blocking and antibody dilution

• Detection specificity:

  • Validate specificity using blocking peptides

  • Include positive controls (e.g., 3T3 cell lysate)

  • Consider SKA2 knockdown or overexpression controls

Validation data shows clear detection in 3T3 cell lysate at 0.5 and 1 μg/ml concentrations, confirming antibody functionality at these recommended dilutions .

How can I optimize co-immunoprecipitation experiments to study SKA2 interactions?

Optimizing co-immunoprecipitation (co-IP) for SKA2 interaction studies:

• Cell lysis conditions:

  • Use mild, non-denaturing lysis buffers (e.g., 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Triton X-100)

  • Include phosphatase inhibitors to preserve phosphorylation-dependent interactions

  • Add protease inhibitors to prevent protein degradation

• Pre-clearing strategy:

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

  • Use species-matched IgG controls to identify non-specific interactions

• Antibody selection and validation:

  • For SKA2 pull-down: validate antibody IP efficiency beforehand

  • For tagged SKA2 constructs: anti-HA or anti-Flag antibodies show good results in SKA2-Sp1 interaction studies

• Specific proven applications:

  • SKA2-GR interactions: confirmed in hippocampal tissue extracts

  • SKA2-Sp1 interactions: successfully demonstrated using Flag-tagged SKA2 and HA-tagged Sp1

  • Use epitope-tagged constructs for improved specificity when antibody cross-reactivity is a concern

• Analysis considerations:

  • Include 5-10% input controls on Western blots

  • Run reciprocal IPs where possible to confirm interactions

What controls should be included when using SKA2 antibodies in research?

Essential controls for SKA2 antibody experiments:

• Specificity controls:

  • Blocking peptide validation: incubate antibody with 5-fold excess (by weight) of immunogenic peptide at 4°C overnight prior to use

  • Knockdown/knockout validation: compare antibody staining in SKA2-depleted vs. control cells

  • Secondary antibody-only control: reveals background from secondary antibody

• Expression manipulation controls:

  • SKA2 overexpression: confirms antibody detection at expected molecular weight

  • siRNA/shRNA-mediated SKA2 knockdown: validates signal reduction with decreased expression

  • Multiple SKA2 siRNAs (e.g., SKA2si3 and SKA2si4) to control for off-target effects

• Application-specific controls:

  • For immunofluorescence: include subcellular markers to validate SKA2's reported kinetochore/spindle localization

  • For Western blot: include loading controls (e.g., β-actin, GAPDH)

  • For reporter assays: include empty vector controls alongside SKA2 overexpression

• Functional validation:

  • Parallel assessment of known SKA2-regulated genes (e.g., PDSS2, FKBP5, SGK1)

  • Molecular phenotypes: cell cycle progression, mitotic checkpoint activation

How can SKA2 antibodies be used to investigate its role in glucocorticoid receptor signaling?

SKA2 antibodies enable multifaceted investigation of SKA2's role in glucocorticoid receptor (GR) signaling:

• Protein-protein interaction studies:

  • Co-immunoprecipitation to detect SKA2-GR complexes in tissue extracts

  • Proximity ligation assays to visualize SKA2-GR interactions in situ

  • ChIP-reChIP to determine co-occupancy at GR target genes

• Subcellular localization:

  • Immunofluorescence to track SKA2-GR co-localization following glucocorticoid treatment

  • Nuclear/cytoplasmic fractionation with Western blot to quantify redistribution

  • Live-cell imaging with fluorescently tagged constructs validated by antibody staining

• Functional analysis:

  • Combine SKA2 antibodies with GR reporter gene assays (e.g., MMTV-Luc) to correlate SKA2 expression with GR activity

  • Monitor GR target gene expression (FKBP5, SGK1, ID3) by RT-PCR following SKA2 manipulation

  • Investigate HSP90-GR heterocomplex composition via sequential co-IPs with SKA2 antibodies

Research has demonstrated that SKA2 enhances GR signaling by counteracting FKBP5 function through interaction with HSP90 cochaperone FKBP4, positioning SKA2 as a positive regulator of GR-mediated glucocorticoid signaling in the central nervous system .

What approaches can resolve contradictory data regarding SKA2's observed molecular weight in Western blots?

The discrepancy between SKA2's calculated molecular weight (14 kDa) and observed weights in Western blots (reported as high as 68 kDa) can be resolved through these methodological approaches:

• Validation strategies:

  • Run parallel samples of recombinant SKA2 protein alongside cell lysates

  • Perform SKA2 knockout/knockdown experiments to identify specific bands that disappear

  • Test multiple antibodies targeting different SKA2 epitopes to identify consistent bands

• Technical modifications:

  • Use gradient gels (4-20%) to better resolve potential SKA2 isoforms

  • Test multiple extraction methods to identify potential protein complexes resistant to denaturation

  • Employ 2D electrophoresis to separate based on both molecular weight and isoelectric point

• Post-translational modification analysis:

  • Treat lysates with phosphatases before Western blot to detect phosphorylation-dependent mobility shifts

  • Test deglycosylation enzymes to identify glycosylated forms

  • Use mass spectrometry to characterize the exact composition of higher molecular weight bands

• Specialized detection methods:

  • Perform immunoprecipitation followed by Western blot under non-reducing and reducing conditions

  • Use antibodies specific to potential SKA2 modifications or isoforms

Understanding these weight discrepancies is critical as they may reflect functionally important modifications or isoforms relevant to SKA2's diverse cellular roles.

How can ChIP-seq experiments be optimized using SKA2 antibodies to identify genomic binding sites?

Optimizing ChIP-seq with SKA2 antibodies requires specialized considerations:

• Antibody validation for ChIP:

  • Test multiple SKA2 antibodies for ChIP efficiency

  • Validate enrichment at known sites (e.g., SKA2-regulated promoters like PDSS2)

  • Perform preliminary ChIP-qPCR before sequencing

  • Include SKA2 knockdown/knockout controls

• Crosslinking optimization:

  • Test different formaldehyde concentrations (0.5-2%)

  • Consider dual crosslinking with disuccinimidyl glutarate (DSG) followed by formaldehyde

  • Optimize crosslinking time (10-15 minutes typically optimal)

• Experimental design considerations:

  • Include input controls and IgG controls

  • Perform biological replicates (minimum of 3)

  • Consider SKA2 ChIP in combination with known interactors (e.g., GR, Sp1)

  • Include treatment conditions relevant to SKA2 function (e.g., dexamethasone)

• Bioinformatic analysis strategy:

  • Perform motif enrichment analysis for transcription factor binding sites (particularly Sp1 sites)

  • Integrate with RNA-seq data following SKA2 knockdown

  • Compare binding patterns with known interactors like GR and Sp1

  • Analyze genomic distribution relative to transcriptional start sites

This approach can help identify genome-wide SKA2 binding patterns and elucidate its transcriptional regulatory functions beyond its established mitotic roles.

What are common issues when working with SKA2 antibodies and how can they be resolved?

How should researchers interpret SKA2 localization data across different experimental conditions?

Interpreting SKA2 localization requires careful consideration of cellular context:

• Cell cycle-dependent localization:

  • Interphase: Predominantly cytoplasmic distribution

  • Mitosis: Concentrated at kinetochores and spindle microtubules

  • Validation: Co-stain with cell cycle markers (e.g., phospho-histone H3)

• Tissue-specific localization patterns:

  • Cytoplasmic localization with GR co-localization in most tissues

  • Nuclear expression specifically observed in breast tumors

  • Interpret: Consider tissue-specific functions beyond mitotic roles

• Experimental perturbation effects:

  • Stress conditions may alter SKA2-GR interactions

  • Dexamethasone treatment can affect SKA2 protein levels in A549 cells

  • Other treatments affecting localization: Staurosporine, phorbol ester, trichostatin A

• Methodological considerations:

  • Fixation artifacts: Compare PFA vs. methanol fixation results

  • Antibody specificity: Verify with multiple antibodies where possible

  • Resolution limitations: Consider super-resolution microscopy for detailed co-localization

• Functional correlations:

  • Compare localization with cell proliferation status

  • Assess relationship between SKA2 localization and mitotic checkpoint activation

  • Correlate localization patterns with GR signaling activity

How can contradictory findings about SKA2 function be reconciled through experimental design?

Resolving contradictory findings about SKA2 function requires systematic experimental approaches:

• Cell type-specific effects:

  • Test identical SKA2 manipulations across multiple cell lines

  • Compare SKA2 interactome between cell types using IP-mass spectrometry

  • Example: SKA2 may have different partners in HepG2 (no detection) vs. A549 cells (high expression)

• Context-dependent functions:

  • Examine SKA2 function under different conditions (normal vs. stress)

  • Test cell cycle-dependent effects through synchronization experiments

  • Example: SKA2's dual roles in mitosis vs. GR signaling require temporal analysis

• Expression level considerations:

  • Titrate SKA2 expression using inducible systems

  • Compare moderate overexpression vs. complete knockout phenotypes

  • Example: SKA2 overexpression increases GC transactivation in HepG2 cells while knockdown in A549 decreases it

• Pathway interaction analysis:

  • Use combinatorial knockdown/overexpression of SKA2 with interacting partners

  • Example: SKA2-FKBP4 relationship where FKBP4 overexpression rescues SKA2 knockdown effects but not vice versa

• In vivo vs. in vitro differences:

  • Compare cell line findings with tissue samples and animal models

  • Develop targeted SKA2 conditional knockout models for tissue-specific analysis

  • Example: SKA2's expression patterns in xenograft models vs. cell lines

How is SKA2 antibody research contributing to our understanding of cancer biology?

SKA2 antibody-based research has revealed several important connections to cancer biology:

• Expression in cancer tissues:

  • Immunohistochemistry with SKA2 antibodies has detected expression in lung and breast tumors

  • SKA2 is highly expressed in hepatocellular carcinoma (HCC) and correlates with tumor stage and patient survival

  • Nuclear SKA2 expression specifically observed in breast tumors, suggesting unique functions

• Molecular mechanisms in cancer:

  • SKA2 represses PDSS2 gene expression through Sp1-binding sites as shown through reporter assays

  • PDSS2 exhibits tumor-suppressing activity independent of its enzymatic function

  • SKA2 knockdown leads to differentially expressed genes involved in cancer pathways

• Clinical correlations:

  • SKA2 expression correlates with tumor staging, tumor grading, patient race, and TP53 mutation status in HCC

  • SKA2 functions within a competing endogenous RNA (ceRNA) network involving SPACA6P-AS/hsa-miR-378a-5p/SKA2, SNHG14/hsa-miR-378a-5p/SKA2, and SNHG15/hsa-miR-378a-5p/SKA2

• Potential therapeutic implications:

  • Combined analysis of SKA2 and PDSS2 expression may serve as prognostic indicators

  • SKA2-promoted malignant phenotypes can be attenuated by PDSS2 overexpression

  • SKA2's involvement in both mitotic progression and gene regulation suggests multiple intervention points

What are the emerging techniques for studying SKA2 using antibody-based approaches?

Emerging antibody-based techniques for studying SKA2:

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM) to visualize SKA2 at kinetochore-microtubule interfaces

  • Live-cell imaging with fluorescently tagged nanobodies against SKA2

  • Correlative light and electron microscopy (CLEM) to resolve SKA2 ultrastructural localization

• Proteomics integration:

  • Proximity-dependent biotin identification (BioID) with SKA2 fusions verified by antibody detection

  • APEX2-based proximity labeling to identify transient SKA2 interactors

  • Multiplexed epitope detection using imaging mass cytometry with SKA2 antibodies

• Chromatin analysis:

  • CUT&RUN or CUT&Tag with SKA2 antibodies for improved chromatin binding profiles

  • HiChIP to identify long-range chromatin interactions at SKA2 binding sites

  • Single-cell ChIP-seq to analyze cell-to-cell variation in SKA2 genomic occupancy

• Clinical applications:

  • Multiplex immunohistochemistry panels including SKA2 for tumor classification

  • Circulating tumor cell analysis with SKA2 antibodies for liquid biopsy applications

  • Development of proximity ligation assays to detect specific SKA2 complexes in patient samples

How can researchers integrate SKA2 antibody studies with genomic and transcriptomic approaches?

Integrative strategies combining SKA2 antibody studies with genomic/transcriptomic approaches:

• Multi-omics experimental design:

  • Parallel ChIP-seq and RNA-seq following SKA2 manipulation

  • Example application: Identified PDSS2 as a downstream target of SKA2 through gene expression profiling after SKA2 knockdown

  • Integrate SKA2 binding data with chromatin accessibility (ATAC-seq) and histone modification maps

• Mechanistic validation workflows:

  • Follow genomic SKA2 binding site identification with:

    • Reporter assays to validate regulatory function

    • CRISPR editing of binding sites to confirm functional importance

    • Protein-DNA interaction verification through EMSA with SKA2 antibodies

  • Example: Validation of SKA2's repression of PDSS2 through luciferase reporter assays with Sp1-binding site mutations

• Clinical data integration:

  • Correlate SKA2 protein levels (by IHC) with transcriptomic profiles in patient cohorts

  • Analyze SKA2 mutations/variants in relation to protein expression and patient outcomes

  • Investigate SKA2's competing endogenous RNA (ceRNA) network identified in HCC

• Single-cell multi-modal analysis:

  • Combine single-cell transcriptomics with antibody-based protein detection

  • Analyze cell cycle-dependent SKA2 functions at single-cell resolution

  • Correlate SKA2 protein levels with mitotic checkpoint activation and cell division outcomes