stac3 Antibody

What is STAC3 and why is it important in research?

STAC3 is a protein containing one phorbol-ester/DAG-type zinc finger and two Src homology 3 (SH3) domains that are essential for mediating interactions with other proteins, especially those that are proline-rich. These structural features enable STAC3 to participate in protein complex assembly vital for various cellular processes, including ion channel regulation and synaptic plasticity . The gene is located on human chromosome 12q13.3, a region associated with several genetic disorders .

In skeletal muscle research, STAC3 is particularly significant as it is a component of the excitation-contraction coupling machinery. It co-localizes with dihydropyridine receptors (DHPRs) and ryanodine receptors (RyR1) at the triad junctions . Recent research has revealed that STAC3 incorporation into skeletal muscle triads can occur independently of the DHPR, suggesting additional binding sites and functions beyond those previously identified .

What types of STAC3 antibodies are commercially available?

Based on the search results, several types of STAC3 antibodies are available for research purposes:

The selection of an appropriate antibody depends on the specific research application, the species being studied, and the detection method employed .

What are the recommended applications for STAC3 antibodies?

STAC3 antibodies have been validated for multiple research applications, with specific methodological considerations for each:

• Western Blotting (WB): Detects STAC3 protein (approximately 42-49 kDa) in tissue or cell lysates. The rabbit polyclonal antibody from Antibodies.com (A28790) is particularly recommended for this application .

• Immunofluorescence (IF): Used to visualize the subcellular localization of STAC3, particularly in muscle triads. For optimal results in myotubes, use the rabbit polyclonal anti-STAC3 at 1:2,000 dilution; for tsA201 cells, use 1:5,000 dilution .

• Immunoprecipitation (IP): Enables isolation of STAC3 protein complexes to study protein-protein interactions. The mouse monoclonal IgG3 kappa antibody (E-2) from Santa Cruz Biotechnology is suitable for this application .

• ELISA: For quantitative measurement of STAC3 protein levels .

For reliable results, antibody specificity should be validated in each experimental system using appropriate controls .

How should samples be prepared for STAC3 immunodetection?

Proper sample preparation is crucial for successful STAC3 detection. For immunofluorescence in cultured cells and tissue sections:

• Cultured myotubes: Fix with paraformaldehyde according to standard protocols. Primary antibodies should be applied at appropriate dilutions (rabbit polyclonal anti-STAC3 at 1:2,000 for myotubes, 1:5,000 for tsA201 cells) .

• Muscle tissue sections: For muscle tissue such as diaphragm, antibodies should be applied at 1:1,000 dilution overnight at 4°C, followed by three washes at 1-hour intervals. Secondary antibodies (e.g., anti-rabbit Alexa 488 at 1:4,000) should be applied for 2 hours at room temperature .

• Imaging parameters: For confocal microscopy, fluorescence can be excited using 488 and 561 nm laser lines and recorded at bandwidths of 493-556 nm (green channel) and 566-752 nm (red channel). Eight-bit images with 1024 × 256 pixels can be acquired at 400 Hz scan speed for optimal visualization of STAC3 localization .

How can researchers verify STAC3 antibody specificity in their experimental systems?

Verifying antibody specificity is essential for reliable research outcomes. The following methodological approaches are recommended:

• Heterologous expression systems: Validate antibody specificity by testing in cells transfected with STAC3 versus related proteins (e.g., STAC1, STAC2). For example, researchers have established antibody specificity in tsA201 cells transiently transfected with either STAC1-GFP, STAC2-GFP, or STAC3-GFP, confirming that anti-STAC3 antibody specifically labeled only STAC3-GFP-transfected cells .

• Knockout/knockdown controls: Where available, tissue or cells lacking STAC3 expression provide excellent negative controls. Western blotting with an antibody generated against Stac3 (recognizing residues 1-63) revealed an approximately 49 kDa protein in wildtype embryos but no protein in mutants, confirming specificity .

• Peptide competition assays: Using blocking peptides such as the STAC3 (E-2) Neutralizing Peptide (sc-514742 P) can confirm binding specificity .

• Cross-reactivity assessment: Test the antibody against tissue samples from different species to confirm the stated species reactivity .

Thorough validation ensures that experimental findings accurately reflect STAC3 biology rather than non-specific interactions or artifacts.

What are the implications of STAC3's triad localization independent of DHPR for experimental design?

Recent research has revealed that endogenous STAC3 can incorporate into triads in the absence of the dihydropyridine receptor (DHPR) in myotubes and muscle fibers of dysgenic mice . This finding contradicts earlier observations using tagged STAC3 constructs and has several important implications for experimental design:

• Endogenous versus tagged protein behavior: Researchers should be cautious when interpreting results from tagged STAC3 constructs, as they may not accurately reflect endogenous protein behavior. In contrast to endogenous STAC3 that localizes to triads independently of DHPR, STAC3-GFP remained diffusely distributed in the cytoplasm of dysgenic myotubes in the absence of a DHPR α1 subunit .

• Triad targeting mechanisms: The unexpected finding indicates that the triad contains binding sites for STAC3 in addition to those identified in the DHPR. This suggests that STAC3 does not merely function as a chaperone for DHPR triad targeting but is independently targeted into the triad .

• Competition between endogenous and exogenous proteins: The putative interaction with endogenous STAC3 appears strong enough to resist competition by heterologously expressed STAC3 constructs . Researchers should consider this when designing overexpression experiments.

• Experimental controls: When studying STAC3 localization, including both DHPR-positive and DHPR-negative (dysgenic) conditions can provide valuable insights into the protein's targeting mechanisms and functions .

These findings suggest that STAC3 plays a more complex role in the excitation-contraction coupling machinery than previously thought, potentially supporting both the functional incorporation of DHPRs into the EC coupling complex and the functional coupling with RyR1 .

What strategies are recommended for co-localization studies involving STAC3?

Co-localization studies are essential for understanding STAC3's interactions with other proteins in the excitation-contraction coupling machinery. Based on the search results, the following methodological approaches are recommended:

• Double immunofluorescence labeling: For co-localization of endogenous STAC3 with other triad proteins such as RyR1:

  • Apply primary antibodies (mouse anti-RyR1 at 1:500 and rabbit STAC3 at 1:1,000) overnight at 4°C

  • Wash three times at 1-hour intervals

  • Incubate with secondary antibodies (anti-mouse Alexa 594 at 1:4,000 and anti-rabbit Alexa 488 at 1:4,000) for 2 hours at room temperature

  • Mount samples in appropriate mounting medium (e.g., Vectashield)

• Confocal microscopy settings: For optimal visualization of co-localization:

  • Use a laser scanning confocal microscope with a high-numerical-aperture oil-immersion lens (e.g., ×63, 1.4 NA)

  • Excite fluorescence using appropriate laser lines (e.g., 488 and 561 nm)

  • Record at specific bandwidths (e.g., 493-556 nm for green channel and 566-752 nm for red channel)

  • Acquire images with appropriate resolution (e.g., 1024 × 256 pixels) at suitable scan speed (e.g., 400 Hz)

• Tagged construct co-localization: When using tagged constructs like GFP-STAC3 or STAC3-HA, design appropriate primers and cloning strategies:

  • For N-terminal tagging (pc-GFP-STAC3): Isolate STAC3 coding sequence by PCR with primers introducing appropriate restriction sites (e.g., SalI and EcoRI)

  • For C-terminal tagging (pc-STAC3-HA): Design primers to introduce appropriate restriction sites and the tag sequence (e.g., KpnI site and HA tag with XhoI site)

• Co-immunoprecipitation: For biochemical confirmation of interactions, co-immunoprecipitation with antibodies against pan-Ry can provide evidence that STAC3 is part of the triadic molecular complex .

These approaches can clarify STAC3's spatial relationships with other proteins and contribute to understanding its role in the excitation-contraction coupling machinery.

How should researchers interpret conflicting data between tagged and endogenous STAC3 localization studies?

The search results reveal an important discrepancy between the localization of tagged STAC3 constructs and endogenous STAC3 in dysgenic myotubes (lacking DHPR) . This conflict highlights several methodological considerations for researchers:

• Recognize the limitations of tagged constructs: Tagged proteins may not always behave identically to their endogenous counterparts. While STAC3-GFP remained diffusely distributed in the cytoplasm of dysgenic myotubes in the absence of a DHPR α1 subunit, endogenous STAC3 showed a clustered distribution colocalized with RyR1 .

• Validate with multiple approaches: When faced with conflicting data, employ multiple detection methods:

  • Use different antibodies targeting different epitopes of STAC3

  • Compare N-terminally tagged (pc-GFP-STAC3) versus C-terminally tagged (pc-STAC3-GFP) constructs

  • Examine localization in both cultured cells and intact tissue

• Consider protein-protein interaction dynamics: The conflicting observations suggest that endogenous STAC3 may interact with additional binding partners at the triad, which could be disrupted or altered by protein tags .

• Examine developmental timing: Compare results in embryonic versus mature tissues. STAC3 has been studied in diaphragm muscle fibers of dysgenic mice at embryonic day E18½, where it organized in transverse double rows of clusters and colocalized with RyR1, similar to wildtype controls .

• Report contradictions transparently: When publishing research, explicitly acknowledge contradictions with previous findings. The authors of search result state: "Here, we report new findings conflicting with these earlier observations."

By carefully considering these factors, researchers can better interpret conflicting data and develop more accurate models of STAC3 function in the excitation-contraction coupling machinery.