PHKA1 Antibody, Biotin conjugated

What is PHKA1 and what is its function in cellular systems?

PHKA1 (Phosphorylase b kinase regulatory subunit alpha, skeletal muscle isoform), also known as Phosphorylase kinase alpha M subunit, plays a crucial role in signal transduction pathways. It functions as a regulatory component of the phosphorylase kinase complex, which catalyzes the phosphorylation of glycogen phosphorylase in response to hormonal and neural signals . This enzyme is particularly important in the regulation of glycogen metabolism in skeletal muscle, converting inactive phosphorylase b to active phosphorylase a, thus facilitating glycogen breakdown and energy production. The protein has been identified with UniProtID P46020, highlighting its established presence in protein databases and research literature .

What are the technical specifications of PHKA1 Antibody, Biotin conjugated?

PHKA1 Antibody, Biotin conjugated is a polyclonal antibody raised in rabbit against human PHKA1. Specifically, the immunogen used in its production is a recombinant human Phosphorylase b kinase regulatory subunit alpha, skeletal muscle isoform protein (amino acids 555-688) . The antibody is of IgG isotype and has been purified using Protein G to achieve >95% purity. It is supplied in liquid form, preserved in a buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 . This particular formulation includes a biotin conjugate, which enhances detection capabilities in various immunoassay applications.

What applications is this antibody validated for?

According to the product information, the PHKA1 Antibody, Biotin conjugated has been validated specifically for ELISA (Enzyme-Linked Immunosorbent Assay) applications . ELISA is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones. The biotin conjugation makes this antibody particularly suitable for ELISA applications as it allows for signal amplification through the strong binding affinity between biotin and streptavidin-HRP conjugates, increasing detection sensitivity. Researchers should note that this antibody has not been explicitly validated for other applications such as Western blotting, immunohistochemistry, or immunofluorescence in the provided data .

What are the proper storage and handling conditions for this antibody?

The PHKA1 Antibody, Biotin conjugated should be stored at -20°C or -80°C upon receipt to maintain its activity and performance . The manufacturer specifically recommends avoiding repeated freeze-thaw cycles, as these can lead to protein denaturation and reduced antibody activity. The antibody is provided in a solution containing 50% glycerol, which helps prevent freeze damage during storage . When handling the antibody, general laboratory precautions should be observed, including wearing gloves and using sterile pipette tips to prevent contamination. For short-term use during experiments, the antibody can be kept on ice, but should be returned to proper storage conditions promptly afterward.

How can I optimize PHKA1 antibody usage in ELISA protocols?

For optimal performance in ELISA applications, several key parameters should be considered when using PHKA1 Antibody, Biotin conjugated:

• Antibody dilution optimization: Perform a titration experiment using serial dilutions (typically 1:500 to 1:5000) to determine the optimal antibody concentration that provides the highest signal-to-noise ratio.

• Blocking optimization: Since the antibody is reactive to human proteins, use a blocking solution containing 3-5% BSA or 5% non-fat milk in PBS to minimize background signal .

• Incubation conditions: For primary antibody binding, incubate at 4°C overnight or at room temperature for 1-2 hours. The biotin conjugation allows for direct detection with streptavidin-HRP without the need for a secondary antibody .

• Detection system: Use a high-quality streptavidin-HRP conjugate and a sensitive substrate such as TMB (3,3',5,5'-Tetramethylbenzidine) for optimal signal development.

• Positive controls: Include recombinant PHKA1 protein or lysates from cell lines known to express PHKA1 to validate antibody performance.

Following these optimization steps will help ensure reliable and reproducible results in your PHKA1 detection experiments.

What are the considerations for using PHKA1 antibody in studying its role in disease models?

When investigating PHKA1 in disease models, particularly in cancer research contexts, several important considerations should be addressed:

• Expression level validation: Recent research indicates that PHKA1-related molecules, such as PHKA1-AS1 (antisense RNA 1), show differential expression between normal and cancer tissues, particularly in non-small cell lung cancer (NSCLC) . Before using the PHKA1 antibody, validate expression levels in your specific disease model using qPCR.

• Signaling pathway context: PHKA1 operates within signal transduction pathways . When studying disease models, consider the broader signaling context and potentially analyze related pathway components simultaneously.

• Cell-type specificity: PHKA1-AS1 has been shown to exhibit different expression patterns across various cell types, with higher expression observed in NSCLC cell lines compared to normal lung epithelial cells . This suggests PHKA1 itself may have cell-type specific functions that should be considered in experimental design.

• Correlation with clinical parameters: Recent studies have correlated PHKA1-AS1 expression with clinical outcomes in cancer patients . Consider designing experiments that can correlate PHKA1 levels with disease progression or patient prognosis.

• Functional validation approaches: Beyond detection, consider using siRNA knockdown or overexpression approaches to validate the functional role of PHKA1, similar to methods used for studying PHKA1-AS1 in recent cancer research .

These considerations will help ensure your research findings have translational relevance and contribute meaningfully to understanding PHKA1's role in disease processes.

How does m6A modification relate to PHKA1 and what techniques can be used to study this connection?

While the provided search results don't directly address m6A modification of PHKA1 itself, they reveal interesting relationships with PHKA1-AS1 that may inform PHKA1 research:

• m6A modification concept: N6-methyladenosine (m6A) is an RNA modification that can affect RNA stability, localization, and function. Recent research has shown that PHKA1-AS1, the antisense RNA related to PHKA1, undergoes m6A modification in NSCLC cells .

• Methodological approaches:

  • m6A-RIP assay: This technique can be used to determine the m6A modification level, as demonstrated with PHKA1-AS1 in NSCLC cell lines compared to normal cells .

  • SRAMP algorithm: This computational tool can predict potential m6A modification sites, as was done for PHKA1-AS1 (identifying sites 26A, 40A, 56A, 182A, 214A, 246A, 322A) .

  • MeRIP coupled qPCR: This method can verify specific m6A modification sites, as shown for PHKA1-AS1 where modifications at 26A, 56A, and 214A were confirmed .

• Functional implications: m6A modification of PHKA1-AS1 was shown to enhance its stability and expression . Researchers investigating PHKA1 might explore whether similar modifications affect PHKA1 mRNA stability and expression levels.

• Role of methyltransferases: METTL3 was identified as an important catalytic enzyme for m6A methylation of PHKA1-AS1 . Similar enzymes might regulate PHKA1 expression and should be considered in comprehensive studies.

Understanding these molecular mechanisms could provide valuable insights for researchers studying PHKA1 regulation and function in both normal and disease contexts.

What controls should I include when using PHKA1 Antibody in experimental protocols?

To ensure experimental rigor and valid interpretation of results, include the following controls when using PHKA1 Antibody, Biotin conjugated:

• Positive controls:

  • Cell/tissue lysates known to express PHKA1 (human skeletal muscle tissues would be appropriate based on PHKA1's function)

  • Recombinant PHKA1 protein, particularly the region corresponding to amino acids 555-688, which was used as the immunogen for this antibody

• Negative controls:

  • Isotype control: Rabbit IgG, biotin-conjugated, to assess non-specific binding

  • Cell lines with PHKA1 knockdown via siRNA or CRISPR (similar to techniques used for PHKA1-AS1 studies)

  • Tissues or cells known not to express PHKA1

• Technical controls:

  • No primary antibody control: Omit the PHKA1 antibody but include all other reagents to assess background

  • Blocking peptide competition: Pre-incubate the antibody with excess immunizing peptide to confirm specificity

  • Concentration gradient: Test different antibody concentrations to determine optimal signal-to-noise ratio

• Validation controls:

  • Correlation with mRNA expression: Perform parallel qPCR to confirm that protein detection correlates with gene expression levels

  • Alternative antibody verification: If possible, use a second PHKA1 antibody targeting a different epitope to confirm results

Inclusion of these controls will substantially strengthen the reliability and interpretability of your experimental findings.

What are the common troubleshooting approaches for PHKA1 antibody experiments?

When encountering issues with PHKA1 Antibody, Biotin conjugated experiments, consider these troubleshooting approaches:

• Weak or No Signal:

  • Verify target expression: Confirm PHKA1 expression in your samples via qPCR

  • Antibody concentration: Increase antibody concentration (decrease dilution)

  • Incubation time/temperature: Extend primary antibody incubation to overnight at 4°C

  • Detection system: Ensure streptavidin-HRP is functional; consider using a more sensitive substrate

  • Sample preparation: Check if protein denaturation conditions are compatible with the epitope recognition

• High Background:

  • Blocking optimization: Increase blocking time or try alternative blocking reagents

  • Washing: Increase washing steps duration and frequency

  • Antibody dilution: Use more diluted antibody solution

  • Buffer composition: Ensure buffer components don't interfere with biotin-streptavidin interaction

• Non-specific Bands/Signals:

  • Increase antibody specificity: Pre-absorb with non-specific proteins

  • Optimize detergent concentration in wash buffers

  • For Western blots: Adjust protein loading and transfer conditions

• Poor Reproducibility:

  • Storage conditions: Ensure proper storage at -20°C or -80°C and avoid repeated freeze-thaw cycles

  • Sample handling: Standardize sample preparation protocols

  • Lot-to-lot variation: Note antibody lot number and test new lots alongside previous ones

• Cross-reactivity Issues:

  • Species validation: Confirm the antibody is suitable for your species (this antibody is specific for human PHKA1)

  • Epitope mapping: Consider potential cross-reactivity with related proteins

These troubleshooting strategies address common issues encountered in antibody-based detection methods and should help optimize your PHKA1 experimental outcomes.

How can I use PHKA1 antibody to investigate protein-protein interactions?

Investigating protein-protein interactions involving PHKA1 requires specialized approaches:

• Co-immunoprecipitation (Co-IP):

  • Use PHKA1 Antibody, Biotin conjugated for pull-down experiments followed by detection of interacting partners

  • Recent research has employed similar techniques to study interactions between PHKA1-AS1 and ACTN4

  • Protocol considerations:
    a. Cross-link biotin-conjugated antibody to streptavidin beads
    b. Prepare cell lysates under non-denaturing conditions to preserve protein interactions
    c. Incubate lysates with antibody-conjugated beads
    d. Wash extensively to remove non-specific binders
    e. Elute and analyze interacting proteins by Western blot or mass spectrometry

• Proximity Ligation Assay (PLA):

  • Combine PHKA1 Antibody, Biotin conjugated with antibodies against suspected interaction partners

  • This technique allows visualization of protein interactions in situ with high specificity

• FRET or BRET Assays:

  • For live cell studies, consider fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) approaches

  • These require additional tagging strategies but provide dynamic interaction information

• Interaction Validation:

  • Based on recent findings with PHKA1-AS1, potential interaction candidates for PHKA1 might include proteins involved in ubiquitination pathways

  • Consider investigating interactions with E3 ubiquitin ligases like SYVN1, which was found to interact with ACTN4 in a system regulated by PHKA1-AS1

• Data Analysis:

  • Quantify interaction strengths under different conditions

  • Compare interactions in normal versus disease states

  • Correlate interactions with functional outcomes

This methodological approach will enable comprehensive characterization of PHKA1's interactome and functional relationships in your experimental system.

What is the optimal protocol for using PHKA1 Antibody in ELISA applications?

ELISA Protocol for PHKA1 Detection:

Materials Required:

• PHKA1 Antibody, Biotin conjugated (50μg or 100μg preparation)

• Coating buffer (0.05M carbonate-bicarbonate buffer, pH 9.6)

• Blocking solution (3% BSA in PBS)

• Wash buffer (0.05% Tween-20 in PBS)

• Streptavidin-HRP conjugate

• TMB substrate solution

• Stop solution (2N H₂SO₄)

• 96-well ELISA plate

Procedure:

• Antigen Coating:

  • Dilute samples or standards in coating buffer

  • Add 100μl per well and incubate overnight at 4°C

  • Wash 3 times with wash buffer

• Blocking:

  • Add 300μl blocking solution to each well

  • Incubate for 1-2 hours at room temperature

  • Wash 3 times with wash buffer

• Primary Antibody:

  • Dilute PHKA1 Antibody, Biotin conjugated to 1:1000 in blocking solution (optimize as needed)

  • Add 100μl per well and incubate for 2 hours at room temperature or overnight at 4°C

  • Wash 5 times with wash buffer

• Detection:

  • Add 100μl streptavidin-HRP (1:5000 in blocking buffer) to each well

  • Incubate for 30 minutes at room temperature

  • Wash 5 times with wash buffer

• Substrate Development:

  • Add 100μl TMB substrate solution to each well

  • Incubate for 15-30 minutes in the dark (monitor color development)

  • Add 50μl stop solution

• Measurement:

  • Read absorbance at 450nm (with 570nm reference if available)

  • Calculate results using standard curve

Notes:

• Considering the antibody's buffer composition (50% Glycerol, 0.01M PBS, pH 7.4), ensure proper dilution to prevent interference

• The storage recommendation to avoid repeated freeze-thaw cycles should be followed for all aliquots

This protocol provides a starting point and should be optimized for specific experimental conditions and sample types.

How can I modify detection methods when using PHKA1 Antibody, Biotin conjugated?

While the PHKA1 Antibody, Biotin conjugated is validated specifically for ELISA applications , researchers may adapt it for other techniques with appropriate modifications:

• Immunohistochemistry (IHC) Adaptation:

  • Dilution range: Start with 1:100-1:500 dilution for optimization

  • Detection system: Use streptavidin-HRP followed by DAB or AEC substrate

  • Antigen retrieval: Test both heat-induced (citrate buffer, pH 6.0) and enzymatic methods

  • Controls: Include skeletal muscle tissue as positive control

• Immunofluorescence (IF) Adaptation:

  • Use fluorophore-conjugated streptavidin (Alexa Fluor 488, 555, or 647) for detection

  • Include DAPI for nuclear counterstaining

  • Set up appropriate filters to detect the specific fluorophore used

  • Consider fixation optimization (4% PFA vs. methanol fixation)

• Flow Cytometry Adaptation:

  • Cell preparation: Use gentle fixation and permeabilization methods

  • Antibody dilution: Start with 1:100 and optimize based on signal intensity

  • Detection: Use fluorophore-conjugated streptavidin

  • Controls: Include unstained cells, isotype controls, and FMO (fluorescence minus one)

• Western Blot Considerations:

  • Sample preparation: Test both reducing and non-reducing conditions

  • Blocking: Use 5% BSA in TBST to minimize biotin background

  • Detection: Employ streptavidin-HRP with enhanced chemiluminescence detection

  • Expected band: Confirm molecular weight of target (~138 kDa for PHKA1)

• Proximity Ligation Assay (PLA) Adaptation:

  • Combine with antibodies against potential interaction partners

  • Follow PLA manufacturer protocols for biotin-conjugated antibodies

  • Optimize primary antibody concentration for best signal-to-noise ratio

These modifications should be carefully validated with appropriate controls before use in critical experiments. Similar methodological approaches have been used successfully with related proteins such as ACTN4 in recent research .

What experimental design is recommended for studying PHKA1 in relation to PHKA1-AS1?

Based on recent research on PHKA1-AS1 and its role in cancer , the following experimental design is recommended for investigating potential relationships between PHKA1 and PHKA1-AS1:

• Expression Correlation Analysis:

  • Use qPCR to quantify both PHKA1 and PHKA1-AS1 expression across:

    • Normal vs. disease tissues (e.g., NSCLC vs. normal lung)

    • Multiple cell lines (e.g., Beas-2b vs. A549, H1299, H1975, PC9, H358, H460)

  • Analyze correlation patterns to determine if their expression is coordinated

• Subcellular Localization Studies:

  • Perform FISH assay for PHKA1-AS1 localization

  • Use immunofluorescence with PHKA1 Antibody (detected via fluorophore-conjugated streptavidin)

  • Assess co-localization patterns in different cellular compartments

• Functional Relationship Assessment:

  • Manipulate PHKA1-AS1 expression (overexpression/knockdown) and measure effects on:

    • PHKA1 protein levels (Western blotting)

    • PHKA1 mRNA levels (qPCR)

    • PHKA1 protein stability (cycloheximide chase assay)

  • Reciprocally, manipulate PHKA1 and assess effects on PHKA1-AS1

• Molecular Interaction Studies:

  • RNA immunoprecipitation (RIP) assay to determine if PHKA1 protein binds to PHKA1-AS1

  • RNA pull-down assay combined with protein spectrum detection to identify proteins that interact with both molecules

• Phenotypic Impact Assessment:

  • Evaluate effects of PHKA1/PHKA1-AS1 manipulation on:

    • Cell proliferation (CCK-8 assay, EdU assay)

    • Cell migration/invasion (wound healing, transwell assays)

    • EMT marker expression (E-cadherin, N-cadherin, Vimentin)

• m6A Modification Analysis:

  • Investigate if PHKA1 mRNA undergoes m6A modification similar to PHKA1-AS1

  • Use m6A-RIP and MeRIP coupled qPCR to identify specific modification sites

  • Assess the role of methyltransferases like METTL3 in regulating both molecules

This comprehensive experimental design will help elucidate the potentially important relationship between PHKA1 and its antisense RNA (PHKA1-AS1) in normal physiology and disease contexts.

How is PHKA1 implicated in cancer research and what methods are recommended for its study?

Recent research suggests important connections between PHKA1-related molecules and cancer, particularly non-small cell lung cancer (NSCLC). While the search results focus primarily on PHKA1-AS1 rather than PHKA1 itself, the findings provide valuable methodological insights for PHKA1 cancer research:

• Expression Analysis in Cancer:

  • PHKA1-AS1 has been found to be highly expressed in NSCLC cells and carcinoma tissues compared to normal controls

  • Similar expression analysis should be conducted for PHKA1 using:

    • qPCR for mRNA quantification across multiple cell lines

    • Western blotting with PHKA1 Antibody for protein level assessment

    • Immunohistochemistry of tissue microarrays containing matched tumor and normal tissues

• Functional Impact Assessment:

  • PHKA1-AS1 has been shown to promote proliferation and metastasis of NSCLC cells

  • To investigate PHKA1's role:

    • Perform gain/loss-of-function studies using overexpression vectors and siRNA approaches

    • Assess effects on cancer hallmarks including proliferation (CCK-8, EdU, colony formation assays), migration and invasion (wound healing, transwell assays)

    • Evaluate EMT marker expression changes (E-cadherin, N-cadherin, Vimentin)

• Mechanistic Investigations:

  • PHKA1-AS1 binds to ACTN4 and inhibits its ubiquitination degradation

  • For PHKA1, investigate:

    • Potential protein-protein interactions using Co-IP approaches

    • Effects on ubiquitination pathways using ubiquitination assays

    • Stability regulation using protein stability assays with cycloheximide chase

• In Vivo Models:

  • PHKA1-AS1 knockdown inhibited lung metastasis in mouse models

  • Similar approaches for PHKA1:

    • Establish xenograft models with PHKA1-manipulated cell lines

    • Use tail vein injection models to assess metastatic potential

    • Analyze tumors using immunohistochemistry for relevant markers

• Clinical Correlation:

  • Analyze PHKA1 expression in patient samples and correlate with:

    • Clinical parameters and outcomes

    • Expression of related molecules (PHKA1-AS1, ACTN4)

    • Potential value as biomarker for diagnosis or prognosis

These methodological approaches, adapted from successful PHKA1-AS1 research, provide a robust framework for investigating PHKA1's potential role in cancer pathogenesis and progression.

What techniques can be used to study PHKA1's role in protein stability and ubiquitination?

Based on findings related to PHKA1-AS1's role in protein stability regulation , the following techniques are recommended to investigate PHKA1's potential involvement in protein stability and ubiquitination processes:

• Protein Stability Assay:

  • Cycloheximide Chase Assay:

    • Treat cells with cycloheximide to inhibit new protein synthesis

    • Collect cells at different time points (0, 2, 4, 6, 8 hours)

    • Analyze protein degradation rates by Western blotting

    • Compare degradation rates between control and PHKA1-manipulated cells

• Proteasomal Degradation Analysis:

  • MG132 Treatment:

    • Treat cells with proteasome inhibitor MG132

    • Analyze if MG132 reverses effects of PHKA1 manipulation on target protein levels

    • This approach can determine if PHKA1-mediated effects involve the proteasomal pathway

• Ubiquitination Detection:

  • Co-Immunoprecipitation (Co-IP) for Ubiquitination:

    • Transfect cells with HA-ubiquitin constructs

    • Treat with MG132 to accumulate ubiquitinated proteins

    • Immunoprecipitate target proteins

    • Detect ubiquitination using anti-HA antibody

    • Compare ubiquitination levels between control and PHKA1-manipulated conditions

• E3 Ligase Interaction Studies:

  • Prediction of E3 Ligases:

    • Use databases like UbiBrowser to predict potential E3 ligases for proteins of interest

  • Validation of E3 Ligase Involvement:

    • Knockdown predicted E3 ligases (e.g., SYVN1, MARCH1, MARCH6)

    • Assess effects on target protein levels

    • Perform Co-IP to validate physical interactions between proteins and E3 ligases

• Binding Studies:

  • RNA Immunoprecipitation (RIP):

    • If PHKA1 potentially interacts with RNA molecules

    • Can be used to assess binding between PHKA1 and potential RNA partners

  • RNA Pull-down:

    • For analyzing protein complexes that may include PHKA1

    • Combined with mass spectrometry for unbiased identification of interaction partners

• Cellular Localization:

  • Subcellular Fractionation:

    • Separate cellular components (cytoplasm, nucleus, membrane)

    • Analyze PHKA1 distribution and co-localization with ubiquitination machinery

  • Immunofluorescence:

    • Visualize PHKA1 localization with respect to proteasomes and target proteins

These techniques, adapted from successful approaches used to study PHKA1-AS1's role in protein stability regulation, provide a comprehensive toolkit for investigating PHKA1's potential involvement in ubiquitination and protein degradation pathways.

How can PHKA1 Antibody be used in multi-parameter analyses?

Advanced multi-parameter analyses with PHKA1 Antibody, Biotin conjugated can provide deeper insights into complex biological systems:

• Multiplex Immunoassays:

  • Combine PHKA1 detection with other biomarkers in multiplex ELISA formats

  • Use differently conjugated antibodies for simultaneous detection of multiple targets

  • Implement microsphere-based multiplex platforms like Luminex

• Multi-Omics Integration:

  • Correlate PHKA1 protein levels (detected via antibody) with:

    • Transcriptomic data (RNA-seq, including PHKA1-AS1 expression)

    • Epigenomic data (m6A modification status)

    • Proteomic data (mass spectrometry)

  • Integrate findings using computational biology approaches

• Spatial Biology Applications:

  • Multiplexed Immunofluorescence:

    • Utilize biotin-streptavidin detection systems with spectrally distinct fluorophores

    • Perform sequential staining with antibody stripping/quenching between rounds

    • Analyze spatial relationships between PHKA1 and other proteins of interest

  • Imaging Mass Cytometry:

    • Label PHKA1 Antibody with metal isotopes

    • Combine with other metal-labeled antibodies for highly multiplexed tissue imaging

• Single-Cell Analysis:

  • Mass Cytometry (CyTOF):

    • Use metal-conjugated streptavidin for detection of biotin-conjugated PHKA1 Antibody

    • Integrate with other cellular markers to identify cell populations with distinct PHKA1 expression patterns

  • Single-Cell Western Blot:

    • Detect PHKA1 protein at single-cell resolution

    • Correlate with other protein markers

• Pathway Analysis:

  • Reverse Phase Protein Array (RPPA):

    • High-throughput analysis of PHKA1 across multiple samples

    • Correlate with other signaling pathway components

  • Phosphorylation State Analysis:

    • Combine with phospho-specific antibodies to correlate PHKA1 expression with pathway activation states

• Dynamic Studies:

  • Live Cell Imaging:

    • Adapt antibody for non-permeabilized detection or use cell-permeable versions

    • Monitor real-time changes in PHKA1 in response to stimuli

  • Time-course Experiments:

    • Analyze PHKA1 expression changes over multiple time points following interventions

These multi-parameter approaches can reveal network-level insights into PHKA1 function and regulation in complex biological systems, particularly in disease contexts like cancer where PHKA1-related molecules have shown significant relevance .

What are the latest methodological advances for studying epitranscriptomic modifications like m6A in relation to PHKA1?

Recent research has highlighted the importance of m6A modification in regulating PHKA1-AS1 , suggesting similar approaches could be valuable for studying PHKA1 regulation:

• High-Throughput m6A Profiling:

  • m6A-seq/MeRIP-seq:

    • Immunoprecipitate m6A-modified RNAs with anti-m6A antibody

    • Perform next-generation sequencing to map modification sites genome-wide

    • Analyze if PHKA1 mRNA contains m6A modifications, similar to findings for PHKA1-AS1

  • miCLIP (m6A individual-nucleotide-resolution crosslinking and immunoprecipitation):

    • Provides single-nucleotide resolution of m6A sites

    • Can identify precise modification sites within PHKA1 transcripts

• Computational Prediction:

  • SRAMP Algorithm:

    • Predict potential m6A modification sites in PHKA1 mRNA

    • Similar approach successfully identified modification sites in PHKA1-AS1 (26A, 40A, 56A, 182A, 214A, 246A, 322A)

  • Integrated Analysis:

    • Combine prediction algorithms with conservation analysis

    • Prioritize sites for experimental validation

• Site-Specific Validation:

  • MeRIP-qPCR:

    • Validate specific m6A sites in PHKA1 transcripts

    • Similar approach confirmed modifications at positions 26A, 56A, and 214A in PHKA1-AS1

  • SELECT (Single-base Elongation and Ligation-based qPCR amplification method):

    • Quantitative analysis of m6A at specific sites

    • Higher specificity than traditional MeRIP-qPCR

• Functional Analysis of m6A Writers/Erasers/Readers:

  • Manipulation of Methyltransferases:

    • Overexpress or knock down METTL3 (shown to regulate PHKA1-AS1)

    • Assess effects on PHKA1 expression and stability

  • Reader Protein Analysis:

    • Investigate binding of YTH domain proteins to potentially m6A-modified PHKA1 transcripts

    • Use RIP assays to confirm interactions

• mRNA Stability Assessment:

  • Actinomycin D Chase Experiments:

    • Treat cells with actinomycin D to inhibit transcription

    • Compare PHKA1 mRNA decay rates under different conditions

    • Similar approach showed METTL3 overexpression slowed PHKA1-AS1 degradation

  • SLAM-seq (Thiol(SH)-Linked Alkylation for the Metabolic sequencing of RNA):

    • Metabolic labeling approach to measure RNA synthesis and decay rates

    • Can determine if m6A affects PHKA1 mRNA half-life

• Translational Impact:

  • Polysome Profiling:

    • Analyze association of PHKA1 mRNA with ribosomes

    • Determine if m6A modification affects translation efficiency

  • Ribosome Profiling:

    • Genome-wide analysis of translation

    • Can reveal if m6A impacts ribosome occupancy on PHKA1 transcripts

These cutting-edge methodological approaches provide powerful tools for investigating the potential epitranscriptomic regulation of PHKA1, building on insights gained from PHKA1-AS1 research .