SUGT1 Antibody

What is SUGT1 and why is it important in biological research?

SUGT1 is a 365-amino acid protein belonging to the SGT1 family that functions as a cochaperone for heat shock protein 90 (HSP90). It plays essential roles in the G1/S and G2/M cell cycle transitions and has dual localization in both cytoplasmic and nuclear compartments. Research interest in SUGT1 has intensified due to its implicated roles in cancer progression, particularly in ovarian cancer where it shows significant overexpression compared to normal tissues . This protein's involvement in cell cycle regulation makes it a critical target for understanding fundamental cellular processes and disease mechanisms.

What types of SUGT1 antibodies are available for research applications?

Researchers can access a diverse array of SUGT1 antibodies, including polyclonal and monoclonal variants that target different epitopes. These antibodies are available in various formats: unconjugated for standard applications, or conjugated with fluorescent tags (Cy3, DyLight488), biotin, or enzymes like HRP for specialized detection methods . When selecting an antibody, researchers should consider target specificity, host species (commonly rabbit or mouse), and whether the antibody targets specific regions (e.g., C-terminal, middle region) as these factors significantly impact experimental outcomes and interpretability.

How do I select the appropriate SUGT1 antibody for my specific research application?

When selecting a SUGT1 antibody, first identify your experimental applications (Western blot, immunofluorescence, flow cytometry, etc.) as antibodies are typically validated for specific techniques. Consider the species reactivity needed—many SUGT1 antibodies react with human, mouse, and rat samples, but cross-reactivity varies significantly between products . Critically evaluate validation data provided by manufacturers, including positive and negative controls. For quantitative applications, monoclonal antibodies generally provide more consistent results, while polyclonal antibodies offer higher sensitivity for detection of low-abundance targets but with potential variability between lots.

What are the optimal conditions for using SUGT1 antibodies in Western blot applications?

For Western blot applications with SUGT1 antibodies, optimal conditions typically include protein denaturation in reducing buffer, separation on 10-12% SDS-PAGE gels, and transfer to PVDF or nitrocellulose membranes. Blocking with 5% non-fat milk or BSA in TBST for 1 hour at room temperature minimizes non-specific binding. For primary antibody incubation, most SUGT1 antibodies perform optimally at dilutions between 1:500 and 1:2000, incubated overnight at 4°C . Include positive controls (cells known to express SUGT1) and negative controls (knockdown cells) to validate specificity. SUGT1 typically appears as a band at approximately 38-41 kDa, though post-translational modifications may alter migration patterns.

How can I optimize SUGT1 antibody performance in immunofluorescence and immunohistochemistry?

For optimal immunofluorescence and immunohistochemistry with SUGT1 antibodies, fixation method is critical—typically 4% paraformaldehyde for 15-20 minutes preserves epitope accessibility while maintaining cellular morphology. Antigen retrieval methods may be necessary, particularly for formalin-fixed, paraffin-embedded tissues, with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) being commonly effective. Permeabilization with 0.1-0.3% Triton X-100 enables antibody access to intracellular SUGT1. Titrate antibody concentrations (typically starting at 1:100-1:500) to optimize signal-to-noise ratio . Given SUGT1's dual cytoplasmic and nuclear localization, counterstaining nuclei with DAPI helps distinguish subcellular distribution patterns when interpreting results.

What are the key considerations for using SUGT1 antibodies in co-immunoprecipitation studies?

For co-immunoprecipitation studies targeting SUGT1 and its binding partners, cell lysis conditions must preserve protein-protein interactions—use non-denaturing buffers containing 0.5-1% NP-40 or CHAPS with protease inhibitors. Pre-clear lysates with protein A/G beads to reduce non-specific binding. When selecting SUGT1 antibodies for immunoprecipitation, prioritize those specifically validated for IP applications . The antibody-to-lysate ratio requires optimization, typically starting with 2-5 μg antibody per 500 μg protein. Include appropriate controls: IgG isotype control to identify non-specific precipitation and input samples (5-10% of lysate) to verify target protein presence before immunoprecipitation. For detecting weak interactions, consider crosslinking approaches to stabilize transient complexes before lysis.

What methods can be employed to investigate SUGT1's interactions with the immune system?

To investigate SUGT1's interactions with the immune system, researchers can employ several sophisticated approaches. Single-sample Gene Set Enrichment Analysis (ssGSEA) has revealed correlations between SUGT1 expression and specific immune cell populations—positively associating with T central memory cells, natural killer cells, and T gamma delta cells, while negatively correlating with activated dendritic cells, cytotoxic T cells, and T helper 1 cells . Flow cytometry using SUGT1 antibodies alongside immune cell markers can validate these computational findings in primary samples. Co-immunoprecipitation followed by immunoblotting can identify direct protein interactions between SUGT1 and immune signaling components. For spatial context, multiplexed immunohistochemistry combining SUGT1 with immune cell markers can reveal proximity relationships within the tumor microenvironment. Functional assays like T cell activation or dendritic cell maturation in the presence of SUGT1 knockdown or overexpression can establish causative relationships.

How do post-translational modifications of SUGT1 affect antibody detection and biological function?

Post-translational modifications (PTMs) of SUGT1 critically influence both antibody detection and biological function. Phosphorylation at specific serine/threonine residues can alter epitope accessibility, potentially reducing antibody binding affinity. When investigating PTMs, researchers should select antibodies targeting regions unlikely to be modified or use modification-specific antibodies. Methodologically, combining immunoprecipitation with phospho-specific Western blotting can reveal regulatory phosphorylation events. Mass spectrometry following SUGT1 immunoprecipitation provides comprehensive PTM profiling, including phosphorylation, ubiquitination, and acetylation patterns. Functionally, these modifications regulate SUGT1's interactions with HSP90 and other chaperone machinery, consequently affecting cell cycle progression. Research designs should incorporate phosphatase inhibitors during sample preparation to preserve physiological modification states and consider using Phos-tag gels to separate differentially phosphorylated SUGT1 forms.

What are common causes of non-specific binding with SUGT1 antibodies and how can these be mitigated?

Non-specific binding with SUGT1 antibodies typically stems from several sources that can be systematically addressed. Insufficient blocking often causes high background—optimize by testing different blocking agents (BSA, normal serum, commercial blockers) at various concentrations (3-5%) and extending blocking times (1-2 hours). Excessive primary antibody concentration leads to non-specific binding; perform careful titration experiments starting with manufacturer recommendations and then testing 2-fold dilution series . Cross-reactivity with structurally similar proteins may occur, particularly with polyclonal antibodies; validate specificity using SUGT1 knockdown/knockout controls alongside wild-type samples. For immunohistochemistry, endogenous peroxidase or phosphatase activity causes background—include appropriate quenching steps (3% hydrogen peroxide for HRP systems). In immunofluorescence, autofluorescence from fixatives can be reduced using sodium borohydride treatment or switching to specific fixation protocols optimized for the tissue type being examined.

How can researchers effectively validate SUGT1 antibody specificity for their experimental systems?

Rigorous validation of SUGT1 antibody specificity requires a multi-faceted approach. Begin with genetic validation using CRISPR/Cas9 knockout or siRNA knockdown systems—a specific antibody will show diminished or absent signal in these negative controls. Peptide competition assays provide another validation method: pre-incubating the antibody with excess immunizing peptide should abolish specific binding. Western blot analysis should reveal a single predominant band at the expected molecular weight (~38-41 kDa for SUGT1) . For tissue studies, compare staining patterns with published literature and RNA expression databases to ensure consistency with expected tissue distribution. Testing multiple antibodies targeting different SUGT1 epitopes can confirm specificity through convergent results. Finally, recombinant SUGT1 can serve as a positive control, particularly when expressed in otherwise negative cell lines, to confirm antibody recognition of the target protein.

What strategies can address inconsistent results between different experimental approaches using SUGT1 antibodies?

Inconsistent results between experimental approaches using SUGT1 antibodies often reflect technique-specific limitations that require systematic troubleshooting. Begin by evaluating whether the antibody is validated for all applications being compared—many antibodies perform well in Western blot but poorly in immunohistochemistry due to epitope accessibility differences . Sample preparation variations significantly impact results; standardize protocols for protein extraction, fixation methods, and buffer compositions across experiments. For quantitative comparisons between techniques, develop standardized positive controls and calibration curves. When comparing results between monoclonal and polyclonal antibodies, recognize that they likely target different epitopes, potentially detecting different SUGT1 isoforms or modified forms. For cell line studies showing discrepancies, confirm SUGT1 expression at the transcript level using qPCR. Consider cell type-specific post-translational modifications that might affect antibody recognition in different experimental contexts, particularly when comparing cell lines to primary tissues.

What methodological approaches can investigate SUGT1's role in cell cycle regulation and its implications for cancer therapy?

Investigating SUGT1's role in cell cycle regulation requires sophisticated methodological approaches with therapeutic implications. Flow cytometry with propidium iodide staining following SUGT1 knockdown or overexpression reveals specific cell cycle arrest patterns (G1/S or G2/M). Time-lapse microscopy with fluorescent cell cycle reporters (FUCCI system) provides dynamic visualization of progression defects in living cells. Co-immunoprecipitation coupled with mass spectrometry can identify cell cycle-specific SUGT1 interaction partners during synchronized cell cycle progression. For therapeutic applications, small molecule screening approaches targeting SUGT1-HSP90 interactions may identify compounds that disrupt this essential chaperone function . High-content imaging following SUGT1 inhibition in combination with established chemotherapeutics can reveal synergistic treatment opportunities. Xenograft models with inducible SUGT1 knockdown systems allow temporal control for assessing SUGT1 inhibition at different treatment stages, providing critical in vivo validation of therapeutic potential.

How can researchers integrate SUGT1 antibody data with bioinformatic analyses to identify novel therapeutic targets?

Integrating SUGT1 antibody data with bioinformatic analyses creates powerful opportunities for therapeutic target discovery. Researchers should begin by correlating protein expression data from immunohistochemistry with transcriptomic profiles to identify co-expressed gene networks—Gene Ontology and KEGG pathway analyses of these networks can reveal biological processes associated with SUGT1 function . Single-cell RNA sequencing combined with SUGT1 protein data can identify cell populations where SUGT1 expression correlates with specific signaling pathways, revealing context-dependent functions. Protein-protein interaction networks constructed from co-immunoprecipitation data can be analyzed for druggable nodes using available databases of small molecule inhibitors. For precision medicine applications, correlate SUGT1 antibody staining patterns with patient treatment responses to identify potential biomarkers for therapy selection. Computational drug repurposing approaches can screen existing compounds for predicted activity against SUGT1-associated pathways, accelerating therapeutic development. Multi-omics integration (combining proteomics, transcriptomics, and epigenomics) provides the most comprehensive understanding of SUGT1's role in disease networks.