Phospho-ACTC1/ACTG1/ACTG2/ACTA1 (Y55/53) Antibody

What are the target proteins of Phospho-ACTC1/ACTG1/ACTG2/ACTA1 (Y55/53) Antibody?

The antibody specifically targets the phosphorylation site Y55/53 in four actin family proteins :

• ACTC1 (alpha cardiac muscle 1): Primarily expressed in cardiac tissue, gene ID P68032

• ACTG1 (cytoplasmic 2/gamma-actin): Ubiquitously expressed, gene ID P63261

• ACTG2 (gamma-enteric smooth muscle): Found in smooth muscle, gene ID P63267

• ACTA1 (alpha skeletal muscle actin): Predominant in skeletal muscle, gene ID P68133

These proteins are highly conserved and involved in diverse cellular functions including contraction, cytoskeletal organization, and cell motility .

What applications is this antibody validated for?

The antibody has been validated for multiple research applications:

The antibody performs consistently across these applications when using the recommended dilutions and protocols .

How should I store and handle this antibody?

For optimal performance and stability:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles as this may degrade antibody quality

• The antibody is provided in liquid form in PBS containing 50% glycerol, 0.5% BSA and 0.02% sodium azide

• Working aliquots can be prepared to minimize freeze-thaw cycles

What is the recommended protocol for Western blot analysis?

For optimal Western blot results with this antibody:

• Protein Extraction and Preparation:

  • Prepare protein lysates in standard lysis buffer

  • Separate proteins by SDS-PAGE using 10% gels

  • Transfer to nitrocellulose membranes using semi-dry transfer

• Antibody Incubation:

  • Block membranes with 5% non-fat milk or BSA

  • Incubate with primary antibody (1:1000 dilution) at 4°C overnight

  • Wash with TBST (3×10 minutes)

  • Incubate with HRP-conjugated secondary antibody (1:2000) for 1 hour

• Detection:

  • Expected molecular weight: 42 kDa for all target proteins

  • The antibody may detect both phosphorylated and hyperphosphorylated isoforms in mitotic cells

How can I validate antibody specificity?

To confirm specificity of phosphorylation detection:

• Phosphatase Treatment Control:

  • Treat cellular extracts with Antarctic phosphatase

  • Western blot analysis should show complete loss of signal after phosphatase treatment

• Mutagenesis Validation:

  • Compare tissues/cells expressing wild-type protein to those expressing Y55/53A mutant (unphosphorylatable)

  • Y55/53A mutants should show no signal with this antibody

• Phosphomimetic Controls:

  • Y55/53E phosphomimetic mutants may be recognized by the antibody similar to wild-type phosphorylated protein

How is actin Y55/53 phosphorylation involved in cellular processes?

Phosphorylation at Y55/53 affects several key processes:

• Cell Cycle Regulation:

  • Similar to serine 406 phosphorylation in HDAC1, Y55/53 phosphorylation may be cell cycle-dependent

  • May be dynamically regulated during mitosis

• Cytoskeletal Dynamics:

  • Affects actin polymerization and F-actin formation

  • Influences interactions with actin-binding proteins

• Cellular Contractility:

  • Y55/53 is positioned near the "A-triad" region of actin, potentially affecting tropomyosin binding and muscle contraction regulation

• Cell Survival:

  • In medulloblastoma, ACTC1 incorporation into F-actin renders cells resistant to apoptosis induced by mitotic inhibitors

What are recommended controls for immunofluorescence experiments?

For reliable immunofluorescence studies:

• Positive Controls:

  • Heart tissue for ACTC1 detection

  • Skeletal muscle for ACTA1

  • Cultured cell lines known to express the phosphorylated targets

• Negative Controls:

  • Secondary antibody only

  • Tissues/cells treated with phosphatase inhibitors

  • Competitive blocking with immunizing peptide

• Visualization Protocol:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Block with 5% BSA

  • Incubate with primary antibody (1:500) overnight at 4°C

  • Use Alexa-Fluor secondary antibodies at 1:500 dilution

  • Co-stain with phalloidin to visualize total F-actin

How can I study the role of actin phosphorylation in cardiac pathologies?

ACTC1 mutations are associated with several cardiac pathologies:

• Experimental Approaches:

  • Compare phosphorylation levels between normal and diseased heart tissues

  • ACTC1 mutations such as p.Gly247Asp have been linked to atrial septal defects and dilated cardiomyopathy

• Functional Analysis:

  • Examine sarcomeric organization using this antibody in combination with other markers

  • Ultrastructural analysis has shown that ACTC1 mutations can lead to sarcomeric disarray and myofibrillar degeneration

  • Perform actin polymerization/depolymerization assays to assess functional impact of phosphorylation

• Animal Models:

  • The antibody has been validated in mouse and rat cardiac tissues

  • Cardiac-specific expression of wild-type vs. mutant ACTC1 can reveal phosphorylation-dependent phenotypes

What is known about kinases that target the Y55/53 site?

While specific kinases targeting Y55/53 are not fully characterized in the search results:

• Candidate Kinases:

  • Tyrosine kinases from Src family are potential candidates

  • Aurora kinases have been implicated in phosphorylating other cytoskeletal proteins during mitosis

• Experimental Approaches:

  • In vitro kinase assays coupled with mass spectrometry

  • Kinase inhibitor screening followed by Western blot with this antibody

  • Kinase overexpression or knockdown experiments to identify responsible enzymes

How can I use this antibody to study actin dynamics in cancer progression?

Research has shown ACTC1 involvement in cancer:

• Medulloblastoma Studies:

  • ACTC1 incorporation into F-actin renders medulloblastoma cells resistant to apoptosis

  • The antibody can be used to track phosphorylation status during cancer progression

• Experimental Design:

  • Compare phosphorylation levels between normal and cancerous tissues

  • Use flow cytometry with this antibody to quantify phosphorylated actin in different cell populations

  • Track changes in phosphorylation after treatment with chemotherapeutic agents

• Functional Assays:

  • Migration assays to correlate phosphorylation with metastatic potential

  • Colony formation assays to assess growth capabilities

  • FACS cell viability assays to measure resistance to apoptosis

What are common issues with phospho-specific antibodies and their solutions?

Common challenges and solutions:

• High Background:

  • Increase blocking time and concentration (5% BSA is often preferable for phospho-antibodies)

  • Optimize antibody dilution (start with manufacturer's recommendation and adjust as needed)

  • Use phosphatase inhibitors in all buffers to preserve phosphorylation status

• Weak or No Signal:

  • Ensure phosphorylation is preserved during sample preparation

  • Use fresh phosphatase inhibitor cocktail in lysis buffers

  • Consider enrichment of phosphorylated proteins using TiO₂ chromatography prior to analysis

  • For IHC applications, test different antigen retrieval methods (citrate buffer pH 6.0 or TE buffer pH 9.0)

• Non-specific Bands:

  • Increase washing stringency

  • Pre-adsorb antibody with non-phosphorylated peptide

  • Validate bands using phosphatase treatment controls

How can I optimize conditions for specific tissue types?

Tissue-specific considerations:

• Cardiac Tissue:

  • ACTC1 is highly expressed in heart tissue

  • For IHC, antigen retrieval with TE buffer pH 9.0 is recommended

  • Include heart tissue as a positive control for ACTC1 detection

• Skeletal Muscle:

  • ACTA1 is predominantly expressed in skeletal muscle

  • More stringent extraction conditions may be needed due to dense sarcomeric structure

• Cell Lines:

  • C2C12 myoblasts can be used for studying ACTC1 expression

  • UW426 and D458 medulloblastoma cell lines have been used successfully with actin antibodies