SIX5 Antibody

What is SIX5 protein and which specific applications are SIX5 antibodies validated for?

SIX5 (SIX homeobox 5) is a transcription factor with a calculated molecular weight of 75 kDa (739 amino acids), although the observed molecular weight in experimental conditions is approximately 100-105 kDa . SIX5 functions as a homeodomain protein involved in gene transcription regulation, with significant implications in developmental processes.

SIX5 antibodies have been extensively validated for multiple research applications, including:

It is important to note that optimal dilutions may be sample-dependent, and researchers should conduct titration experiments to determine optimal conditions for their specific experimental system .

What are the recommended storage conditions for maintaining SIX5 antibody stability?

For optimal stability and performance, SIX5 antibodies should be stored at -20°C . Under these conditions, these antibodies typically remain stable for one year after shipment . The standard formulation includes PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which contributes to stability during storage .

Important stability considerations:

• Aliquoting is generally unnecessary for -20°C storage

• Some commercial preparations (20μl sizes) contain 0.1% BSA as a stabilizing agent

• The standard rabbit IgG formulations include sodium azide (0.02%) as a preservative

• Avoid repeated freeze-thaw cycles to maintain antibody performance

How do researchers validate SIX5 antibody specificity for experimental applications?

Validation of SIX5 antibodies involves multiple complementary approaches to confirm specificity and reliability:

• Orthogonal validation: Comparing antibody-based results with independent non-antibody-based methods to verify target detection .

• Independent antibody validation: Using two or more antibodies directed toward different epitopes of SIX5 to confirm staining pattern consistency .

• Cell line validation: Testing antibody performance across multiple cell lines with known SIX5 expression (e.g., A549, HeLa, Y79) .

• siRNA knockdown: Evaluating the decrease in antibody-based staining intensity upon SIX5 downregulation to confirm specificity .

• Tagged protein comparison: Assessing signal overlap between antibody staining and GFP-tagged SIX5 protein expression .

Comprehensive validation results in scores such as "Enhanced," "Supported," "Approved," or "Uncertain," depending on the degree of validation and performance consistency across multiple assays .

How do SIX5 mutations impact its functionality in developmental disorders?

SIX5 mutations have been implicated in Branchio-oto-renal syndrome (BOR), an autosomal dominant developmental disorder characterized by branchial arch defects, hearing loss, and other developmental abnormalities . Several missense mutations have been identified in patients with BOR, including A158T, A296T, G365R, and T552M .

Functional analyses of these mutations have revealed two primary mechanisms of pathogenicity:

• Disruption of protein-protein interactions: Some mutations affect the ability of SIX5 to bind to its interaction partner EYA1, which is also implicated in BOR syndrome .

• Impaired transcriptional activation: Mutations can disrupt the ability of SIX5 or the EYA1-SIX5 complex to activate gene transcription, thereby affecting downstream developmental processes .

For investigating these mutations, researchers have successfully employed:

• Yeast two-hybrid assays to assess protein-protein interactions

• PCR-based mutagenesis to introduce patient-derived mutations into expression constructs

• Cell culture-based transcriptional activation assays

These approaches provide valuable insights into the molecular mechanisms underlying SIX5-associated developmental disorders .

What are the critical factors to consider when performing immunohistochemistry (IHC) with SIX5 antibodies?

When performing IHC with SIX5 antibodies, several critical factors can significantly impact results:

How can researchers address discrepancies between theoretical and observed molecular weights of SIX5?

• Post-translational modification analysis: SIX5 may undergo various post-translational modifications (phosphorylation, glycosylation, etc.) that increase its apparent molecular weight. Researchers can:

  • Use phosphatase treatment to remove phosphorylation

  • Apply deglycosylation enzymes to remove glycosylation

  • Compare migration patterns before and after treatments

• Alternative splicing investigation: Different isoforms may be expressed in different tissues or cell types. Researchers should:

  • Compare expression patterns across multiple cell lines/tissues

  • Use isoform-specific primers for RT-PCR validation

  • Consider sequencing to confirm isoform identity

• Validation with multiple antibodies: Using antibodies targeting different epitopes can help confirm that the higher molecular weight band is indeed SIX5 and not a cross-reactive protein .

• Protein loading and separation optimization:

  • Use gradient gels (e.g., 4-15%) for better separation of higher molecular weight proteins

  • Optimize running conditions (time, voltage) for proteins >100 kDa

  • Include appropriate molecular weight markers spanning 50-150 kDa

What are the most effective approaches for optimizing immunoprecipitation (IP) protocols with SIX5 antibodies?

Successful immunoprecipitation of SIX5 requires careful optimization:

• Antibody quantity determination: The recommended range is 0.5-4.0 μg of SIX5 antibody per 1.0-3.0 mg of total protein lysate . Optimal antibody amount should be determined empirically by testing multiple concentrations.

• Lysis buffer selection: Consider the subcellular localization of SIX5 (nuclear transcription factor):

  • Use nuclear extraction buffers or whole-cell lysis buffers with nuclear disruption capabilities

  • Include appropriate protease and phosphatase inhibitors

  • Optimize salt concentration to maintain protein-protein interactions of interest

• Pre-clearing strategy: To reduce non-specific binding:

  • Pre-clear lysates with protein A/G beads

  • Use control IgG from the same species as the SIX5 antibody

  • Optimize pre-clearing time (typically 1-2 hours)

• Washing stringency optimization: Balance between:

  • Higher stringency (higher salt concentration) to reduce non-specific binding

  • Lower stringency to maintain specific interactions

  • Typically start with 3-5 washes using progressively less stringent buffers

• Elution method selection:

  • Denaturing (SDS buffer with heating) for maximum yield

  • Non-denaturing (peptide competition) for maintaining protein activity

  • Include appropriate controls to validate the specificity of precipitated proteins

For co-immunoprecipitation studies investigating SIX5-EYA1 interactions, these optimizations are particularly critical .

How can SIX5 antibodies be utilized to study its role in transcriptional regulation networks?

SIX5 functions as a homeodomain transcription factor regulating various genes, and antibodies can be powerful tools for investigating its regulatory networks:

• Chromatin immunoprecipitation (ChIP): SIX5 antibodies can be used to:

  • Identify genomic binding sites through ChIP-seq

  • Validate predicted binding sites from computational analyses

  • Study dynamic binding patterns across different cell types or conditions

  • Investigate co-occupancy with other transcription factors

• Co-immunoprecipitation coupled with mass spectrometry: This approach allows:

  • Identification of protein interaction partners in the transcriptional complex

  • Detection of condition-specific interactions

  • Discovery of novel regulatory mechanisms

• Immunofluorescence co-localization: Using SIX5 antibodies alongside antibodies for other transcription factors to:

  • Visualize nuclear localization and potential co-localization with other factors

  • Examine dynamics of nuclear entry/exit under different conditions

  • Correlate with transcriptional activity using nascent RNA labeling

• Proximity ligation assays (PLA): To detect and quantify:

  • Direct protein-protein interactions in situ

  • Changes in interaction frequencies under different conditions

  • Spatial distribution of interactions within the nucleus

These approaches can be particularly valuable for understanding SIX5's role in regulating genes like DMPK and ATP1A1, and how disruptions in these regulatory networks may contribute to disorders like DM1 (myotonic dystrophy type 1) .

What are the emerging applications of SIX5 antibodies in developmental biology and disease research?

SIX5 antibodies are increasingly being applied in several cutting-edge research areas:

• Developmental pathway analysis:

  • Tracing SIX5 expression during embryonic development

  • Correlating expression patterns with developmental milestones

  • Investigating interactions with other developmental regulators like EYA1

• Disease mechanism investigations:

  • Studying the role of SIX5 in Branchio-oto-renal syndrome (BOR) pathogenesis

  • Examining potential implications in other developmental disorders

  • Investigating connections to myotonic dystrophy through DMPK regulation

• Single-cell applications:

  • Integration with single-cell RNA-seq data to correlate protein expression with transcriptional profiles

  • Cellular heterogeneity analysis in development and disease

  • Application in human tissue atlases to map cell type-specific expression

• Therapeutic target validation:

  • Evaluating SIX5 as a potential therapeutic target in developmental disorders

  • Studying the effects of potential modulators on SIX5 function

  • Developing screening assays for compounds that might rescue mutant SIX5 function

These emerging applications highlight the importance of highly specific and well-validated SIX5 antibodies in advancing our understanding of fundamental developmental processes and disease mechanisms.