PI4KG1 Antibody

What is the target specificity of PI4KG1 Antibody?

The PI4KG1 Antibody specifically binds to the 230 kDa PI4K protein, which catalyzes the phosphorylation of phosphatidylinositol to produce phosphatidylinositol 4-phosphate (PI4P). This molecule serves as a crucial precursor for important signaling lipids including PIP2 and PIP3, which regulate numerous cellular processes. When designing experiments, it's important to consider this specificity, particularly when working with tissues known to express high levels of PI4K, such as brain tissue and certain cancer cell lines.

Which experimental applications is PI4KG1 Antibody validated for?

The PI4KG1 Antibody has been rigorously validated for several key experimental techniques:

It is advisable to optimize these dilutions for your specific experimental system to achieve optimal results .

How should PI4KG1 Antibody be stored to maintain its efficacy?

For optimal performance, store the PI4KG1 Antibody at -20°C in its recommended buffer. The antibody typically remains stable for one year after shipment when stored properly. For antibodies in this class, the storage buffer typically contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is generally unnecessary for -20°C storage, but may be advisable if the antibody will be accessed frequently to avoid freeze-thaw cycles that could degrade the protein structure.

How can PI4KG1 Antibody be used to investigate mitochondrial dynamics?

Recent studies have utilized PI4KG1 Antibody to elucidate PI4K's involvement in mitophagy, where its activity has been linked to PINK1-PRKN signaling. To apply this in your research:

• Experimental Design: Compare PI4P levels in wild-type versus PINK1 knockout cells using PI4KG1 for detection

• Methodology:

  • Treat cells with mitochondrial uncouplers (e.g., CCCP at 10 μM for 3 hours)

  • Fix and permeabilize cells using 4% paraformaldehyde and 0.1% Triton X-100

  • Incubate with PI4KG1 Antibody (1:500 dilution)

  • Co-stain with mitochondrial markers such as TOM20

  • Analyze by confocal microscopy and quantify colocalization

This approach allows researchers to visualize reduced PI4P levels in PINK1 knockout cells, indicating impaired mitochondrial quality control mechanisms.

What are the considerations for using PI4KG1 Antibody in cancer biology research?

The PI4KG1 Antibody has proven valuable in profiling PI4K expression across cancer cell lines, revealing its potential role in promoting cell proliferation via lipid metabolism. When designing cancer research experiments:

• Cell Line Selection: Include both PI4K-overexpressing and normal expression cell lines for comparison

• Controls: Implement RNAi knockdown controls to validate antibody specificity

• Complementary Techniques: Combine antibody-based detection with:

  • PI4P lipid measurements (using lipid extraction and mass spectrometry)

  • Cell proliferation assays correlating PI4K levels with growth rates

  • Pathway inhibition experiments targeting downstream effectors

Researchers should be aware that PI4K expression can vary significantly between cancer types, necessitating thorough baseline characterization before intervention studies.

How can cross-reactivity be addressed when working with PI4KG1 Antibody?

When investigating PI4K in complex systems, addressing potential cross-reactivity is essential:

• Validation Approaches:

  • Perform parallel experiments with a second antibody targeting a different PI4K epitope

  • Include genetic controls (knockdown or knockout) to confirm specificity

  • Conduct peptide competition assays to verify binding specificity

• Data Analysis:

  • When performing quantitative analyses, subtract background signals obtained from knockout samples

  • Use recombinant PI4K protein standards to establish detection sensitivity and linearity ranges

  • Consider ratiometric approaches comparing PI4K signal to housekeeping proteins

These rigorous validation steps ensure that observed signals truly represent PI4K activity rather than cross-reactive proteins with similar epitopes.

What strategies can improve weak or inconsistent signals when using PI4KG1 Antibody in Western blots?

When encountering signal issues with PI4KG1 Antibody:

• Sample Preparation Optimization:

  • Ensure complete lysis by using stronger buffers containing 1% NP-40 or RIPA buffer

  • Include phosphatase inhibitors to preserve phosphorylated forms

  • Load sufficient protein (typically 30-50μg for cellular PI4K detection)

• Detection Enhancement:

  • Increase antibody concentration incrementally (start with 1:1000 and adjust if needed)

  • Extend primary antibody incubation time to overnight at 4°C

  • Use more sensitive detection systems such as enhanced chemiluminescence plus (ECL+)

• Technical Considerations:

  • Ensure efficient transfer of high molecular weight proteins (230 kDa) by using:

    • Lower percentage gels (6-8%)

    • Extended transfer times or high-molecular-weight transfer protocols

    • Addition of 0.1% SDS to transfer buffer

  • Block with 5% BSA instead of milk if detecting phosphorylated forms

For consistent results, perform a preliminary titration experiment to determine optimal antibody concentration for your specific samples .

How can specificity be validated when using PI4KG1 for immunoprecipitation studies?

To ensure PI4KG1 Antibody specificity in immunoprecipitation:

• Pre-clearing Strategy:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Pre-incubate antibody with beads before adding to lysate

• Control Experiments:

  • Perform parallel IPs with non-immune rabbit IgG

  • Include a PI4K-depleted sample as negative control

  • Verify pulled-down complexes by multiple detection methods:

    • Western blot with a different PI4K antibody

    • Mass spectrometry validation of precipitated proteins

    • Functional assays measuring kinase activity of the immunoprecipitate

• Optimization Parameters:

  • Adjust antibody-to-lysate ratio (recommended 0.5-4.0 μg antibody per 1-3 mg lysate)

  • Test different lysis buffers to preserve protein interactions

  • Consider crosslinking the antibody to beads to prevent IgG contamination in eluates

These approaches will help ensure that immunoprecipitated complexes accurately represent PI4K interactions rather than non-specific binding .

How can PI4KG1 Antibody be integrated into multi-omics research approaches?

For researchers pursuing integrated multi-omics studies:

• Proteomics Integration:

  • Use PI4KG1 for immunoprecipitation followed by mass spectrometry to identify interaction partners

  • Combine with phosphoproteomics to map downstream signaling pathways affected by PI4K activity

  • Develop targeted proteomics assays for PI4K using the epitope information from the antibody

• Lipidomics Correlation:

  • Correlate PI4K protein levels (detected by PI4KG1) with PI4P quantification by lipidomics

  • Establish causality through PI4K inhibition or activation followed by lipid profile analysis

  • Map spatial distribution of PI4K and its lipid products through combined immunofluorescence and lipid imaging

• Transcriptomics Complement:

  • Compare PI4K protein levels with mRNA expression to identify post-transcriptional regulation

  • Design experiments to capture transcriptional changes downstream of PI4K signaling

This integrated approach provides a comprehensive understanding of PI4K's role within cellular networks and its impact on lipid metabolism pathways.

What considerations apply when using PI4KG1 Antibody in tissue microarray (TMA) analyses?

When applying PI4KG1 Antibody to tissue microarray studies:

• Protocol Optimization:

  • For optimal antigen retrieval, use TE buffer at pH 9.0 (similar to PI4KA protocols)

  • Alternative option: citrate buffer at pH 6.0 if initial results are suboptimal

  • Titrate antibody concentration starting at 1:50-1:500 dilution range for IHC applications

• Control Implementation:

  • Include known positive tissues (brain tissue shows reliable PI4K expression)

  • Incorporate negative control tissues with minimal PI4K expression

  • Add on-slide antibody controls (primary antibody omission, isotype controls)

• Analysis Approach:

  • Develop quantitative scoring systems accounting for both staining intensity and percentage of positive cells

  • Consider automated image analysis for objective quantification

  • Validate findings with orthogonal methods such as RNAscope for mRNA detection

These methodological considerations ensure robust and reproducible results when using PI4KG1 Antibody in large-scale tissue studies .

How might PI4KG1 Antibody contribute to understanding PI4K's role in neurodegenerative diseases?

Given PI4K's expression in brain tissue, PI4KG1 Antibody could significantly advance neurodegenerative disease research:

• Experimental Applications:

  • Compare PI4K localization and activity in post-mortem tissues from patients with neurodegenerative conditions versus controls

  • Analyze PI4K association with disease-specific protein aggregates using co-immunoprecipitation

  • Investigate PI4K dynamics in cellular and animal models of neurodegeneration

• Methodological Approach:

  • Perform double immunofluorescence with PI4KG1 and markers of cellular stress or protein aggregation

  • Utilize super-resolution microscopy to examine subcellular PI4K distribution in neurons

  • Develop brain region-specific PI4K activity assays validated by antibody-based detection

• Translational Potential:

  • Correlate PI4K levels or activity with disease progression biomarkers

  • Evaluate PI4K as a potential therapeutic target in neurodegeneration

  • Develop screening assays for compounds that modulate PI4K function

This research direction could reveal novel connections between phosphoinositide signaling and neurodegenerative processes .

What are the considerations for using PI4KG1 Antibody in studying drug resistance mechanisms in cancer?

Cancer researchers investigating drug resistance mechanisms should consider:

• Experimental Design Strategy:

  • Compare PI4K expression and localization between drug-sensitive and resistant cell lines

  • Monitor PI4K activity during acquisition of resistance using longitudinal sampling

  • Combine PI4KG1 Antibody detection with phosphoinositide measurements to correlate protein expression with activity

• Methodological Considerations:

  • Develop cell line panels with varying degrees of drug resistance

  • Implement PI4K knockdown or overexpression to establish causality in resistance mechanisms

  • Use PI4KG1 Antibody in combination with other pathway markers to map resistance networks

• Data Analysis Framework:

  • Correlate PI4K expression levels with IC50 values for various therapeutics

  • Perform network analysis of PI4K-interacting proteins in resistant versus sensitive cells

  • Develop predictive models incorporating PI4K status as a biomarker of treatment response

This application of PI4KG1 Antibody could reveal novel resistance mechanisms and potential combination therapy approaches targeting phosphoinositide signaling.