PHKA1 Antibody, HRP conjugated

What is PHKA1 and what cellular functions does it mediate?

PHKA1 (phosphorylase kinase, alpha 1) is a muscle-specific regulatory subunit of phosphorylase kinase, an enzyme complex essential for glycogen metabolism. The protein functions primarily by catalyzing the phosphorylation of serine residues in specific substrates, including troponin I . PHKA1 has a calculated molecular weight of approximately 137 kDa, though it typically appears as bands at 130-140 kDa in Western blot applications . The alpha chain of the protein has been demonstrated to bind calmodulin, suggesting regulatory functions beyond phosphorylation activity . PHKA1 is predominantly expressed in skeletal muscle tissues, with significant expression also detected in heart tissue, as confirmed by multiple immunodetection methods . Recent research has also identified a long non-coding RNA associated with PHKA1 (PHKA1-AS1) that appears to be upregulated in certain cancer types, notably non-small cell lung cancer (NSCLC) .

What are the primary applications for HRP-conjugated PHKA1 antibodies?

HRP-conjugated PHKA1 antibodies are versatile research tools optimized for various immunodetection techniques. The primary applications include:

• Western Blotting (WB): HRP-conjugated PHKA1 antibodies provide excellent visualization in Western blot applications with recommended dilutions typically ranging from 1:500 to 1:1000 . The HRP conjugate enables direct chemiluminescent detection without requiring secondary antibody incubation.

• ELISA: These antibodies demonstrate high sensitivity in enzyme-linked immunosorbent assays, offering quantitative detection of PHKA1 protein .

• Immunohistochemistry (IHC): Though less common with direct HRP conjugates, modified protocols allow for tissue section analysis, particularly with paraffin-embedded samples requiring antigen retrieval .

• Immunofluorescence (IF): While fluorescent conjugates are typically preferred for IF applications, HRP-conjugated antibodies can be used with appropriate substrate systems for chromogenic detection .

The HRP enzyme facilitates detection through its ability to catalyze reactions that produce either colored, chemiluminescent, or fluorescent products when appropriate substrates are added, with the specific detection method determined by the experimental requirements and available imaging systems .

What are the recommended storage conditions for maintaining PHKA1 antibody activity?

For optimal preservation of antibody functionality, HRP-conjugated PHKA1 antibodies should be stored at -20°C in appropriate buffer conditions. The standard storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, the antibodies remain stable for at least one year after shipment, making aliquoting unnecessary for -20°C storage for most research applications .

For smaller volume preparations (20μl), manufacturers often include 0.1% BSA as a stabilizing protein to prevent loss of activity during freeze-thaw cycles . It is important to note that sodium azide, while an effective preservative for the antibody protein, can inhibit HRP activity at higher concentrations; therefore, researchers should ensure dilution to working concentrations before application in enzymatic detection systems . Following manufacturer guidelines is essential, as formulations may vary slightly between suppliers, potentially affecting long-term stability and performance characteristics.

What are the optimal dilution ratios for different experimental applications?

Optimization of dilution ratios is critical for achieving specific signal detection while minimizing background. Based on validated protocols, the following application-specific dilutions are recommended:

These recommendations should serve as starting points for optimization, as the optimal dilution can vary based on sample type, protein expression level, and detection system sensitivity . For skeletal muscle tissue samples, where PHKA1 is highly expressed, higher dilutions may provide sufficient signal, while samples with lower expression levels may require more concentrated antibody solutions . It is strongly recommended that each new antibody lot be titrated in specific experimental systems to obtain optimal results, as batch-to-batch variations can affect optimal working concentrations .

What sample preparation techniques ensure optimal PHKA1 detection?

Effective sample preparation is crucial for reliable PHKA1 detection using HRP-conjugated antibodies. For tissue samples, particularly skeletal muscle where PHKA1 is predominantly expressed, several considerations are important:

• For Western blotting: Complete cell lysis is essential, typically using RIPA or NP-40 based buffers supplemented with protease inhibitors. Samples should be homogenized thoroughly and cleared by centrifugation before determining protein concentration .

• For immunohistochemistry: Paraffin-embedded human skeletal muscle tissue requires appropriate antigen retrieval. Heat-mediated antigen retrieval with citrate buffer pH 6.0 is generally recommended, though some protocols suggest using TE buffer at pH 9.0 as an alternative . Complete deparaffinization and rehydration before antigen retrieval are essential steps.

• For immunofluorescence: Cell fixation methods significantly impact epitope accessibility. For HeLa cells, where positive IF/ICC detection has been validated, paraformaldehyde fixation (4%) followed by permeabilization with 0.1-0.5% Triton X-100 provides reliable results .

When working with muscle tissues specifically, the high protein density and complex architecture may require extended incubation times with primary antibodies to ensure adequate penetration and specific binding . Additionally, blocking with 5% BSA or normal serum matching the secondary antibody host species helps reduce background signal in all applications .

How can researchers troubleshoot non-specific binding issues with PHKA1 antibodies?

Non-specific binding is a common challenge when working with antibodies, including HRP-conjugated PHKA1 antibodies. Several methodological approaches can mitigate these issues:

• Optimize blocking conditions: Increase blocking reagent concentration (5-10% BSA or normal serum) and extend blocking time to 1-2 hours at room temperature. For particularly problematic samples, overnight blocking at 4°C may be beneficial .

• Adjust antibody dilution: Higher dilutions often reduce non-specific binding. Begin with the manufacturer's recommended range and increase dilution if background persists while maintaining specific signal .

• Modify washing procedures: Increase the number and duration of wash steps using PBS-T (PBS with 0.05-0.1% Tween-20). For Western blots, at least four wash cycles of 5-10 minutes each are recommended .

• Include competing proteins: Addition of 0.1-0.5% BSA or 5% non-fat dry milk to antibody diluents can reduce non-specific interactions while preserving specific epitope recognition .

• Validate antibody specificity: Use appropriate controls such as PHKA1 knockout cell lysates, which have been successfully employed to confirm antibody specificity in Western blot applications. The absence of signal at the expected 130 kDa in knockout samples confirms specificity .

If background issues persist despite these optimizations, pre-absorption of the antibody with the immunogen peptide (when available) can significantly reduce non-specific binding, though at the cost of potentially reducing specific signal intensity as well .

How does PHKA1 expression vary across different tissue types and what implications does this have for experimental design?

PHKA1 exhibits distinct tissue-specific expression patterns that significantly impact experimental design considerations. Based on validated antibody detection methods:

• High expression tissues: PHKA1 shows strongest expression in skeletal muscle tissue, with substantial expression also detected in heart tissue . This tissue-specific distribution reflects its primary role in glycogen metabolism regulation in contractile tissues.

• Moderate expression: Certain cell lines, including HeLa cells, demonstrate detectable PHKA1 expression suitable for immunofluorescence studies .

• Variable expression: Western blot analyses across multiple cell lines show differential expression patterns:

  • Human skeletal muscle lysates consistently show strong bands at the expected 130-140 kDa range

  • A431 and Jurkat cell lysates show detectable but variable expression

  • HeLa cells exhibit moderate expression levels when analyzed by Western blotting

These expression patterns necessitate careful experimental planning. When studying tissues with low PHKA1 expression, researchers should consider:

• Using lower antibody dilutions (1:50-1:200 range)

• Employing enhanced detection systems with higher sensitivity

• Increasing sample loading for Western blot applications

• Extending primary antibody incubation times to improve signal detection

Conversely, in high-expression tissues like skeletal muscle, standard protocols with recommended dilutions typically yield robust signals, potentially allowing for higher antibody dilutions to conserve reagents while maintaining specific detection .

What are the key considerations when using HRP-conjugated PHKA1 antibodies in multiplex detection systems?

Multiplex detection involving HRP-conjugated PHKA1 antibodies requires careful experimental design to ensure specific signal isolation and prevent cross-reactivity:

• Enzymatic detection limitations: Standard HRP detection systems are challenging to use in traditional multiplex settings due to the difficulty in distinguishing between multiple HRP signals. To overcome this:

  • Consider sequential detection protocols with HRP inactivation between rounds

  • Utilize HRP substrates with different colorimetric outputs when possible

  • Employ tyramide signal amplification (TSA) with spectrally distinct fluorophores

• Antibody compatibility: When using PHKA1 antibodies alongside other primary antibodies:

  • Select antibodies raised in different host species to enable species-specific secondary detection

  • Verify that epitope regions do not overlap with other target proteins

  • Consider potential cross-reactivity with similar kinase family members

• Optimized loading control detection: For Western blot applications, researchers have successfully used anti-PHKA1 antibody [EPR12118] in combination with mouse anti-Alpha Tubulin [DM1A] as a loading control. This combination allows visualization of the 130 kDa PHKA1 band distinct from the 50 kDa tubulin band .

• Signal separation strategies: For optimal results with multiplex fluorescent Western blotting:

  • PHKA1 detection with 800CW secondary antibody

  • Loading control detection with 680RD secondary antibody

  • This combination minimizes spectral overlap and enables clear signal discrimination

These strategies facilitate simultaneous detection of PHKA1 alongside other proteins of interest while maintaining specificity and reducing experimental variability.

How do post-translational modifications of PHKA1 affect antibody recognition and experimental outcomes?

Post-translational modifications (PTMs) of PHKA1 can significantly influence antibody epitope recognition, potentially affecting experimental outcomes in various applications:

• Phosphorylation effects: As a regulatory subunit of phosphorylase kinase, PHKA1 itself undergoes phosphorylation as part of its functional regulation. Antibodies targeting regions containing phosphorylation sites may show differential binding depending on the phosphorylation status . This is particularly relevant when studying:

  • Active versus inactive enzyme states

  • Signaling pathway analyses

  • Metabolic regulation studies

• Epitope accessibility: Different antibody clones target specific regions of the PHKA1 protein:

  • Antibodies targeting amino acids 555-688 (regulatory subunit A region) have been validated for multiple applications

  • Antibodies recognizing the C-terminal region (AA 631-730) demonstrate effectiveness in Western blot, immunofluorescence, and immunohistochemistry applications

  • Antibodies directed against amino acids 828-1062 provide broader application compatibility

The accessibility of these epitopes may be affected by protein conformation changes induced by PTMs, protein-protein interactions, or sample preparation methods.

• Application-specific considerations: Researchers should be aware that:

  • Denaturing conditions in Western blotting may expose epitopes normally masked in native confirmation

  • Fixation methods for immunohistochemistry can differentially preserve or alter PTM status

  • Live-cell applications require consideration of dynamic PTM states

When studying PHKA1 regulation or activity-dependent changes, researchers may benefit from using multiple antibodies targeting distinct epitopes to provide complementary information about the protein's modification state and functional status .

What is known about PHKA1 dysregulation in pathological conditions and how can antibodies aid in these investigations?

PHKA1 dysregulation has been implicated in several pathological conditions, where HRP-conjugated antibodies serve as valuable tools for investigating disease mechanisms:

• Glycogen Storage Diseases: Mutations in PHKA1 are associated with X-linked muscle glycogenosis (glycogen storage disease type IX), characterized by exercise intolerance and myopathy. HRP-conjugated antibodies enable detection of altered protein expression or localization in patient-derived samples, facilitating both diagnostic and mechanistic studies .

• Cancer Biology: Recent research has identified significant associations between PHKA1-related molecules and cancer progression:

  • PHKA1-AS1 (antisense long non-coding RNA) shows elevated expression in non-small cell lung cancer (NSCLC) cells and tissues compared to normal controls

  • Quantitative PCR analysis demonstrated that PHKA1-AS1 expression was significantly higher in NSCLC cell lines (A549, H1299, H1975, PC9, H358, and H460) compared to normal lung epithelial cells (Beas-2b)

  • Analysis of 23 NSCLC patient samples revealed consistently higher PHKA1-AS1 expression in carcinoma tissues compared to adjacent non-cancerous tissues

• Muscle Disorders: As a muscle-specific protein involved in energy metabolism, PHKA1 alterations may contribute to various myopathies. Immunohistochemical analysis using PHKA1 antibodies on skeletal muscle tissue enables evaluation of protein distribution patterns and potential abnormalities in diseased states .

For disease-focused investigations, researchers should consider selecting antibodies targeting disease-relevant epitopes and combining immunodetection with functional assays to correlate protein expression patterns with physiological outcomes .

How can PHKA1 antibodies be applied in RNA-protein interaction studies related to PHKA1-AS1?

Recent research highlighting the importance of PHKA1-AS1 (the antisense long non-coding RNA associated with PHKA1) in cancer biology opens new avenues for investigation where PHKA1 antibodies can play crucial roles:

• RNA Immunoprecipitation (RIP) applications: HRP-conjugated PHKA1 antibodies can be adapted for RIP protocols to investigate potential physical interactions between PHKA1 protein and PHKA1-AS1 or other RNAs. This approach requires:

  • Optimization of cross-linking conditions to preserve RNA-protein complexes

  • Modification of standard immunoprecipitation protocols to maintain RNA integrity

  • Implementation of RNase inhibitors throughout the experimental workflow

• Co-localization studies: Combining PHKA1 antibody detection with RNA fluorescence in situ hybridization (FISH) for PHKA1-AS1 enables visualization of potential spatial relationships between the protein and its antisense transcript. This dual-detection approach can provide insights into regulatory mechanisms and functional relationships in normal and pathological states .

• Regulatory pathway investigations: In NSCLC models, where PHKA1-AS1 shows elevated expression, PHKA1 antibodies can be used to:

  • Assess whether altered PHKA1-AS1 expression affects PHKA1 protein levels through immunoblotting

  • Evaluate changes in PHKA1 subcellular localization through immunofluorescence

  • Monitor PHKA1 post-translational modifications potentially influenced by PHKA1-AS1

These applications require careful consideration of antibody specificity and may benefit from using multiple detection techniques to provide comprehensive insights into the complex relationships between PHKA1 protein function and its associated regulatory RNAs in health and disease contexts .

What are the most effective strategies for validating PHKA1 antibody specificity?

Rigorous validation of PHKA1 antibody specificity is essential for generating reliable research data. Multiple complementary strategies have proven effective:

• Knockout/knockdown validation: The gold standard approach involves comparing detection in wild-type versus PHKA1-deficient samples:

  • Western blot analysis using PHKA1 knockout HEK-293T cell lysates has successfully demonstrated antibody specificity by showing absence of the expected 130 kDa band in knockout samples while maintaining clear detection in wild-type samples

  • This approach provides definitive confirmation of antibody specificity under denaturing conditions

• Multi-tissue panel validation: Leveraging PHKA1's known tissue expression pattern provides an effective validation strategy:

  • Detecting strong signals in skeletal muscle and heart tissue (known high-expression tissues)

  • Observing weaker or absent signals in tissues with lower expression

  • This pattern verification across multiple tissues confirms expected specificity profiles

• Peptide competition assays: Pre-incubating the antibody with excess immunizing peptide should substantially reduce or eliminate specific binding:

  • The resulting signal reduction in Western blot, IHC or IF applications provides evidence of epitope-specific recognition

  • This approach is particularly valuable when knockout models are unavailable

• Multiple antibody concordance: Using antibodies targeting different PHKA1 epitopes:

  • Consistent detection patterns across different antibodies (targeting distinct regions such as AA 555-688 versus AA 631-730) provides supporting evidence for specificity

  • Discrepancies between antibodies may indicate epitope-specific factors affecting detection

Implementation of these validation strategies, particularly the knockout validation approach, significantly enhances confidence in experimental results and should be considered essential for publications employing PHKA1 antibodies .

What protocol modifications optimize detection of low-abundance PHKA1 in non-muscle tissues?

Detecting PHKA1 in tissues with lower expression levels than skeletal muscle requires specific protocol optimizations to enhance sensitivity while maintaining specificity:

• Sample preparation enhancements:

  • Implement protein concentration methods such as immunoprecipitation before Western blotting

  • For tissue sections, extend antigen retrieval times (20-30 minutes) with citrate buffer (pH 6.0) or TE buffer (pH 9.0)

  • Consider using fresh-frozen samples rather than formalin-fixed when possible for better epitope preservation

• Signal amplification strategies:

  • Employ tyramide signal amplification (TSA) systems which can increase detection sensitivity 10-100 fold over standard HRP-based methods

  • Utilize polymer-HRP detection systems which provide multiple HRP molecules per binding event

  • Consider extended substrate incubation times with lower substrate concentrations to build signal while minimizing background

• Antibody protocol adjustments:

  • Use lower dilutions (1:50-1:100 for IHC/IF; 1:200-1:500 for Western blot)

  • Extend primary antibody incubation to overnight at 4°C

  • Implement repeated antibody application with intermittent washing for difficult samples

• Detection optimization:

  • For Western blot applications, use high-sensitivity chemiluminescent substrates with extended exposure times

  • For microscopy-based detection, increase exposure settings while ensuring control samples remain in the linear detection range

  • Consider confocal microscopy with signal averaging for IF applications to improve signal-to-noise ratio

These modifications have successfully enabled detection of PHKA1 in cell lines with relatively lower expression, such as A431 and Jurkat cells, which show detectable but significantly reduced expression compared to skeletal muscle tissue .