PHKA1 Antibody

What is PHKA1 and what is its physiological function?

PHKA1 (phosphorylase kinase alpha 1) is a gene that encodes the alpha subunit of the phosphorylase b kinase enzyme found predominantly in muscle cells. The enzyme is a complex structure composed of 16 subunits (four each of alpha, beta, gamma, and delta subunits). The alpha subunit specifically helps regulate the activity of phosphorylase b kinase .

Physiologically, phosphorylase b kinase plays a crucial role in cellular energy provision through glycogen metabolism. It activates glycogen phosphorylase b by converting it to the more active form, glycogen phosphorylase a, which then breaks down glycogen into glucose for energy utilization, particularly during exercise .

What applications are PHKA1 antibodies validated for?

PHKA1 antibodies have been validated for multiple research applications:

Most PHKA1 antibodies have been tested with human, mouse, and rat samples, with observed molecular weights typically around 130-140 kDa, compared to the calculated 137 kDa .

What are the recommended protocols for optimizing PHKA1 antibody detection in Western blotting?

For Western blot optimization with PHKA1 antibodies:

• Sample preparation: Use skeletal muscle lysates as positive controls, as they show strong PHKA1 expression. Human skeletal muscle, mouse skeletal muscle, and mouse heart tissue lysates have demonstrated reliable detection .

• Loading amount: Optimal results have been reported with 10-20 μg of total protein per lane .

• Antibody dilution: Start with 1:1000 dilution for recombinant monoclonal antibodies or 1:500-1:1000 for polyclonal antibodies .

• Expected band size: Look for bands at approximately 130-140 kDa. Some researchers report observing the protein at 130 kDa rather than the calculated 137 kDa .

• Validation approach: For confirmation of specificity, PHKA1 knockout cell lysates have been used successfully. The knockout-tested antibodies show no signal at the expected molecular weight in PHKA1 knockout HEK-293T cell lysates, confirming specificity .

What antigen retrieval methods are recommended for PHKA1 immunohistochemistry?

Two main antigen retrieval methods have shown efficacy for PHKA1 detection in IHC:

• Citrate buffer method:

  • pH 6.0 citrate buffer heat-mediated antigen retrieval prior to IHC staining protocol .

  • This method has been validated for paraffin-embedded human skeletal muscle tissue.

• TE buffer method:

  • pH 9.0 TE buffer is suggested as primary antigen retrieval method .

  • Alternatively, citrate buffer pH 6.0 can be used if TE buffer doesn't yield optimal results.

The choice between these methods may depend on tissue type, fixation method, and specific antibody clone. For tissues with high collagen content (like skeletal muscle), the higher pH method (TE buffer pH 9.0) often provides better antigen retrieval .

How should researchers troubleshoot non-specific binding with PHKA1 antibodies?

When encountering non-specific binding with PHKA1 antibodies, researchers should consider:

• Blocking optimization:

  • For Western blot: Use 5% milk in TBS-0.1% Tween® 20 (TBS-T) for blocking membranes .

  • For IHC/IF: Extend blocking time with serum-based blockers.

• Antibody specificity validation:

  • Use PHKA1 knockout controls where available (e.g., PHKA1 knockout HEK-293T cells) .

  • Consider orthogonal RNA-seq validation to correlate antibody signals with mRNA expression levels .

• Cross-reactivity assessment:

  • Be aware that PHKA1 is most abundantly expressed in skeletal muscle, with lower expression in other tissues.

  • Non-specific bands in non-muscle tissues might indicate cross-reactivity with other phosphorylase kinase subunits.

• Secondary antibody optimization:

  • For fluorescence-based detection, Goat anti-Rabbit IgG H&L 800CW has been validated at 1/20000 dilution .

  • For chemiluminescence, titrate secondary antibodies to minimize background.

How can PHKA1 antibodies be used to study glycogen storage diseases?

PHKA1 mutations are associated with glycogen storage disease type IX (GSD IXd or X-linked muscle glycogenosis), making PHKA1 antibodies valuable tools for studying this condition :

• Diagnostic approach:

  • Immunohistochemistry of muscle biopsies can reveal abnormal PHKA1 expression patterns.

  • Western blot analysis can quantify PHKA1 protein levels, which may be reduced in patients with GSD IXd.

• Functional studies:

  • Combine PHKA1 antibody detection with enzymatic activity assays to correlate protein levels with phosphorylase kinase activity.

  • Immunoprecipitation with PHKA1 antibodies can help isolate the enzyme complex for further functional analysis.

• Patient-derived samples:

  • When analyzing patient muscle biopsies, use antigen retrieval with TE buffer pH 9.0 for optimal IHC results .

  • Compare PHKA1 expression with glycogen accumulation patterns in affected muscle tissues.

• Research considerations:

  • Some patients with PHKA1 mutations show no clinical symptoms despite biochemical abnormalities, suggesting compensatory mechanisms that can be explored using PHKA1 antibodies in conjunction with other metabolic markers .

What is the significance of PHKA1-AS1 in cancer research, and how can PHKA1 antibodies contribute to this field?

Recent research has identified PHKA1-AS1, a long non-coding RNA, as highly expressed in non-small cell lung cancer (NSCLC) and potentially involved in cancer progression :

• Expression correlation studies:

  • PHKA1 antibodies can be used to determine whether PHKA1-AS1 expression correlates with PHKA1 protein levels in cancer cells.

  • The impact of PHKA1-AS1 on PHKA1 protein expression can be assessed through Western blot analysis after manipulating PHKA1-AS1 levels.

• Mechanistic investigations:

  • Research has shown that PHKA1-AS1 promotes NSCLC cell migration, invasion, and proliferation .

  • PHKA1 antibodies can help elucidate whether these effects involve changes in PHKA1 protein function or downstream glycogen metabolism alterations.

• Experimental approach:

  • After PHKA1-AS1 knockdown or overexpression, researchers can use PHKA1 antibodies (1:1000 dilution for Western blot) to assess changes in protein expression .

  • Immunofluorescence with PHKA1 antibodies (1:10-1:100 dilution) can reveal changes in subcellular localization in cancer cells .

How can researchers differentiate between different phosphorylase kinase subunits in experimental settings?

Phosphorylase kinase is composed of multiple subunits (α, β, γ, and δ), making specific detection challenging:

• Antibody selection:

  • Choose antibodies raised against unique epitopes of PHKA1. For example, antibody ab176338 was generated using a recombinant immunogen specific to PHKA1 .

  • Antibodies targeting the immunogen sequence "AHSLRCSAEEATEGLMNLSPSAMKNLLHHILSGKEFGVERSVRPTDSNVSPAISIHEIGAVGATKTERTGIMQLKSEIKQSPGTSMTPSSGSFPSAYDQQSSKDSRQGQWQ" have shown specificity for PHKA1 .

• Validation techniques:

  • Western blot analysis using knockout cell lines (PHKA1 knockout HEK-293T cells) provides definitive confirmation of specificity .

  • Compare band patterns in tissues with known differential expression of phosphorylase kinase subunits (e.g., skeletal muscle vs. liver).

• Multiple detection methods:

  • Combine immunodetection with mass spectrometry for unambiguous identification of phosphorylase kinase subunits.

  • Use RT-PCR to correlate protein detection with mRNA expression of specific subunits.

What are the optimal sample preparation methods for detecting PHKA1 in different tissue types?

Sample preparation varies by tissue type and experimental application:

• Skeletal muscle tissue:

  • For Western blot: Homogenize in RIPA buffer with protease inhibitors. Load 10 μg of protein for optimal detection .

  • For IHC: Fix in 10% neutral buffered formalin, process to paraffin, and section at 4-6 μm. Use TE buffer pH 9.0 for antigen retrieval .

• Cell lines:

  • For Western blot: Cell lines such as A431, Jurkat, HeLa, and HEK-293T have been successfully used. Lyse cells in RIPA buffer and load 10-20 μg protein per lane .

  • For flow cytometry: Permeabilize HeLa cells and use 1:10 dilution of PHKA1 antibody for intracellular staining .

• Protein stability considerations:

  • When studying protein stability, researchers can treat cells with cycloheximide (CHX, 100 µg/mL) to inhibit protein synthesis and track PHKA1 degradation over time .

  • For proteasome involvement studies, MG132 (10 µM) treatment can be used in conjunction with PHKA1 antibody detection .

What controls should be included when working with PHKA1 antibodies?

Proper controls are essential for reliable PHKA1 antibody experiments:

• Positive controls:

  • Skeletal muscle tissue lysates (human or mouse) show robust PHKA1 expression .

  • Cell lines with confirmed PHKA1 expression include A431, Jurkat, HeLa, and HEK-293T (wild-type) .

• Negative controls:

  • PHKA1 knockout cell lines (e.g., PHKA1 knockout HEK-293T) provide definitive negative controls .

  • For IHC/IF, omitting primary antibody while maintaining secondary antibody incubation helps identify non-specific secondary antibody binding.

• Loading controls:

  • For Western blot normalization, alpha-tubulin has been validated (Anti-alpha Tubulin antibody [DM1A] at 1/20000 dilution) .

• Antibody specificity controls:

  • For flow cytometry, comparison with rabbit IgG (negative) has been used successfully .

  • For novel applications, pre-absorption with the immunizing peptide can confirm specificity.

How can researchers optimize detection of PHKA1 in immunofluorescence studies?

Successful immunofluorescence detection of PHKA1 requires attention to several parameters:

• Cell fixation and permeabilization:

  • For HeLa cells (where PHKA1 detection has been validated), 4% paraformaldehyde fixation followed by permeabilization is recommended .

  • The FISH assay protocol, which includes 4% paraformaldehyde fixation and specific buffer treatments (A, C, E, F), has been used successfully for subcellular localization studies .

• Antibody dilution:

  • Start with 1:10-1:100 dilution range for immunofluorescence applications .

  • For intracellular flow cytometry, 1:10 dilution has been validated .

• Signal amplification:

  • For tissues with lower PHKA1 expression, consider using tyramide signal amplification to enhance detection sensitivity.

  • Confocal microscopy provides better resolution for subcellular localization studies compared to conventional fluorescence microscopy .

• Nuclear counterstaining:

  • DAPI (4', 6-diamidino-2-phenylindole) has been successfully used as a nuclear counterstain in PHKA1 immunofluorescence studies .

What are the challenges in studying PHKA1 function in disease models?

Researchers face several challenges when investigating PHKA1 in disease contexts:

• Expression variability:

  • PHKA1 expression is predominantly in skeletal muscle, with lower levels in other tissues, making detection challenging in non-muscle samples .

  • Some patients with PHKA1 mutations show no clinical symptoms despite biochemical abnormalities, suggesting complex regulatory mechanisms .

• Enzyme complex dynamics:

  • PHKA1 functions as part of a large 16-subunit enzyme complex, making it difficult to isolate its specific contribution to enzymatic activity.

  • The alpha subunit is known to bind calmodulin, adding another layer of regulatory complexity .

• Tissue-specific isoforms:

  • Different tissues express different phosphorylase kinase subunit combinations (e.g., muscle vs. liver), requiring careful experimental design and antibody selection .

• Future research opportunities:

  • Investigating the relationship between PHKA1-AS1 (long non-coding RNA) and PHKA1 protein function in cancer .

  • Exploring the potential role of PHKA1 in other metabolic disorders beyond glycogen storage diseases.

How does m6A modification affect PHKA1-AS1, and what methodological approaches can be used to study this?

Recent research has identified m6A modification of lncRNA PHKA1-AS1 as enhancing Actinin Alpha 4 (ACTN4) effects in cancer :

• Experimental approach:

  • RNA stability assays using actinomycin D (5 µg/mL) treatment followed by qPCR analysis can assess how m6A modification affects PHKA1-AS1 stability .

  • Transfection with METTL3 plasmid (an m6A methyltransferase) followed by PHKA1-AS1 expression analysis has been used to investigate this relationship .

• Combined protein-RNA studies:

  • After manipulating m6A modification (e.g., via METTL3 overexpression), researchers can use PHKA1 antibodies to determine if changes in PHKA1-AS1 affect PHKA1 protein expression.

  • Western blotting for PHKA1 (1:500-1:1000 dilution) can be combined with qPCR for PHKA1-AS1 to establish correlation .

• Methodological considerations:

  • RNA extraction should avoid degradation to maintain lncRNA integrity.

  • qPCR primer design must ensure specificity for PHKA1-AS1 without amplifying the PHKA1 mRNA.

  • For studying protein stability in relation to RNA changes, cycloheximide chase assays (CHX, 100 µg/mL) have been validated .