pi4k2b Antibody

What is PI4K2B and what cellular functions does it regulate?

PI4K2B (phosphatidylinositol 4-kinase type 2 beta) is an enzyme that catalyzes the phosphorylation of phosphatidylinositol to generate phosphoinositide lipids. These lipids are crucial for regulating membrane trafficking, signal transduction, and cytoskeletal dynamics . PI4K2B is primarily localized in the cytosol but can also be found in endosomes, plasma membrane, and trans-Golgi network . The enzyme plays a significant role in controlling trafficking from the trans-Golgi network to endosomes through the recruitment of clathrin adaptors . Recent research has revealed that PI4K2B is involved in early T cell activation and may function as a suppressor of cell invasion in some contexts .

What applications are PI4K2B antibodies commonly used for?

PI4K2B antibodies are versatile tools used in multiple experimental applications:

Researchers should optimize antibody dilutions for each specific application and sample type to achieve optimal results .

How should PI4K2B antibodies be stored to maintain reactivity?

For optimal stability and performance, PI4K2B antibodies should be:

• Stored at -20°C in aliquots to avoid repeated freeze-thaw cycles that can degrade antibody quality

• Maintained in buffers typically containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Used within their specified validity period, typically 6-12 months

Maintaining proper storage conditions is critical as antibody degradation can lead to reduced sensitivity and non-specific binding in experimental applications. For long-term storage, creating small aliquots is recommended to prevent repeated freeze-thaw cycles of the entire stock .

Which host species and sample types work best with PI4K2B antibodies?

Most commercial PI4K2B antibodies are:

• Developed in rabbits as polyclonal antibodies

• Reactive primarily with human samples, though many also detect mouse and rat PI4K2B

• Successfully tested on various sample types including cell lines (HepG2, 293T, HeLa, MCF-7, A549), tissue lysates (liver, kidney), and biological fluids (serum, plasma)

When working with less common species or sample types, preliminary validation experiments are recommended to confirm cross-reactivity before proceeding with full-scale experiments .

What are the optimal conditions for Western blot detection of PI4K2B?

For successful Western blot detection of PI4K2B:

• Sample preparation:

  • Use 30 μg of protein per lane under reducing conditions

  • Common positive controls include HepG2, 293T, HeLa, and MCF-7 cell lysates

• Electrophoresis conditions:

  • Run on 5-20% SDS-PAGE gel at 70V (stacking)/90V (resolving) for 2-3 hours

• Transfer and detection:

  • Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

  • Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

  • Incubate with primary antibody (0.5-2 μg/mL) overnight at 4°C

  • Wash with TBS-0.1% Tween (3 times, 5 minutes each)

  • Incubate with appropriate HRP-conjugated secondary antibody (typically 1:5000)

  • Develop using an enhanced chemiluminescent detection system

The expected band size for PI4K2B is approximately 50-55 kDa .

What is the recommended protocol for immunofluorescent detection of PI4K2B?

For optimal immunofluorescent detection:

• Cell preparation:

  • Fix cells using an appropriate fixative (e.g., paraformaldehyde)

  • Permeabilize cells to allow antibody access to intracellular targets

• Antigen retrieval (for tissue sections):

  • For FFPE samples, use heat-induced epitope retrieval with citrate buffer (pH 6.0) or TE buffer (pH 9.0)

  • For enzyme antigen retrieval, incubate for 15 minutes with appropriate retrieval reagent

• Staining procedure:

  • Block with 10% goat serum to reduce non-specific binding

  • Incubate with PI4K2B antibody (5 μg/mL) overnight at 4°C

  • Wash thoroughly with PBS

  • Incubate with fluorophore-conjugated secondary antibody (e.g., Cy3-conjugated anti-rabbit at 1:100 dilution) for 30 minutes at 37°C

  • Optional counterstaining with nuclear dyes or cytoskeletal markers (e.g., Phalloidin-iFluor 488)

  • Mount and visualize using appropriate fluorescence microscope filters

A431 and HepG2 cells have been successfully used as positive controls for immunofluorescence applications .

How can I quantify PI4K2B protein levels in serum or plasma samples?

For quantitative analysis of PI4K2B in biological fluids:

• ELISA is the preferred method:

  • Sandwich ELISA kits are available with detection ranges of 0.313-20 ng/mL

  • Minimum detection limit is typically around 0.188-0.313 ng/mL

• Sample preparation:

  • Collect serum or plasma according to standard protocols

  • Avoid hemolyzed samples as they may interfere with assay results

  • Centrifuge samples after collection to remove particulates

• Assay procedure:

  • Follow the manufacturer's protocol for the specific ELISA kit

  • Maintain consistent incubation times and temperatures

  • Run standards in duplicate or triplicate for accurate quantification

  • Create a standard curve using the provided PI4K2B standards

• Data analysis:

  • Calculate PI4K2B concentration from the standard curve

  • Account for any sample dilution factors

  • Consider stability issues (loss rate <5% within expiration date under appropriate storage)

The assay is typically performed in a 96-well pre-coated plate format and uses colorimetric detection methods .

How can PI4K2B antibodies be used to study its role in cancer progression?

PI4K2B has been implicated as a potential tumor suppressor, and antibodies can be powerful tools to investigate this role:

• Expression analysis:

  • Compare PI4K2B protein levels between normal and cancerous tissues/cells using Western blot or IHC

  • Correlate expression levels with cancer stage, invasion potential, or patient prognosis

  • Mining oncogenomic databases has revealed that loss of the PI4K2B allele and underexpression of PI4K2B mRNA are associated with human cancers of epithelial origin

• Functional studies:

  • Use PI4K2B antibodies to validate knockdown efficiency in siRNA experiments

  • Research has shown that PI4K2B depletion can confer an aggressive invasive phenotype on minimally invasive cell lines like HeLa and MCF-7

  • Examine matrix degradation capability using fluorescent gelatin degradation assays after PI4K2B depletion

• Mechanistic investigations:

  • Study co-localization of PI4K2B with endosomal markers (Rab5, Rab7) or exocytic markers (Rab8) using dual immunofluorescence

  • Investigate MT1-MMP trafficking following PI4K2B knockdown, as PI4K2B has been shown to maintain MT1-MMP traffic in the degradative pathway

  • Monitor changes in actin cytoskeleton organization, as PI4K2B depletion affects this structure

These approaches can help elucidate the molecular mechanisms by which PI4K2B suppresses cancer invasion and potentially identify new therapeutic targets .

What methodological approaches can resolve discrepancies in PI4K2B localization studies?

Researchers occasionally report conflicting results regarding PI4K2B subcellular localization. To resolve such discrepancies:

• Employ multiple detection methods:

  • Combine biochemical fractionation with immunoblotting

  • Use super-resolution microscopy techniques for more precise localization

  • Consider live-cell imaging with fluorescently tagged PI4K2B to observe dynamic localization patterns

• Address antibody specificity concerns:

  • Validate antibody specificity using PI4K2B knockout or knockdown controls

  • Compare results with multiple antibodies targeting different epitopes of PI4K2B

  • Check for cross-reactivity with related PI4K family members

• Consider experimental conditions affecting localization:

  • Cell type differences (PI4K2B localization may vary between cell types)

  • Cell cycle stage (phosphoinositide metabolism changes throughout the cell cycle)

  • Growth conditions and cellular stress that may alter PI4K2B distribution

• Investigate isoform-specific localization:

  • Distinguish between PI4KIIα and PI4KIIβ isoforms, which show distinct localization patterns

  • Use isoform-specific antibodies or tagged constructs

Research has shown that while PI4KIIα and PI4KIIβ both synthesize PI(4)P, they create separate pools with distinct functions in TGN-to-endosome traffic .

How can I design experiments to study PI4K2B's role in phosphoinositide signaling pathways?

To investigate PI4K2B's specific contributions to phosphoinositide signaling:

• Use PI(4)P reporter systems:

  • Utilize the PI(4)P reporter glutathione S-transferase (GST) fused to the PH domain of FAPP1 to visualize PI(4)P pools

  • Compare PI(4)P distribution after selectively inhibiting different PI4K isoforms

  • Wortmannin can be used to inhibit PI4KIII activities while studying PI4KII-generated pools

• Manipulate PI4K2B activity:

  • Design siRNA experiments to deplete PI4K2B specifically

  • Use CRISPR/Cas9 for gene knockout studies

  • Compare phenotypes with those observed after depleting other PI4K family members to identify unique functions

• Analyze downstream effectors:

  • Monitor recruitment of clathrin adaptors that are dependent on PI4K2B-generated PI(4)P

  • Investigate effects on membrane trafficking pathways

  • Assess changes in TGN-to-endosome transport

• Study kinase activity directly:

  • Perform in vitro kinase assays using immunoprecipitated PI4K2B

  • Analyze phosphorylation of specific substrates

  • Measure PI(4)P production using sensitive detection methods

These approaches can help distinguish the specific roles of PI4K2B-generated PI(4)P from those produced by other kinases in the complex phosphoinositide signaling network .

What are the best approaches for multiplexed detection of PI4K2B and interacting partners?

For comprehensive analysis of PI4K2B interaction networks:

• Co-immunoprecipitation strategies:

  • Use PI4K2B antibodies to pull down protein complexes

  • Analyze by mass spectrometry to identify novel binding partners

  • Validate interactions by reciprocal co-immunoprecipitation and Western blotting

• Proximity labeling approaches:

  • Generate BioID or APEX2 fusion proteins with PI4K2B

  • Identify proteins in close proximity through biotinylation

  • Combine with PI4K2B antibody validation of identified partners

• Multiplexed immunofluorescence:

  • Perform dual or triple immunofluorescence with antibodies to PI4K2B and suspected partners

  • Use appropriate combinations of primary antibodies from different host species

  • Select non-overlapping fluorophores for secondary antibodies

  • Analyze co-localization using confocal microscopy and quantitative image analysis

• FRET-based interaction studies:

  • Utilize fluorescently tagged PI4K2B and interaction partners

  • Measure FRET signals to confirm direct interactions

  • Validate interactions using PI4K2B antibodies in complementary assays

These methods can reveal how PI4K2B participates in protein complexes that regulate membrane trafficking and signaling pathways .

What are common issues when using PI4K2B antibodies and how can they be resolved?

Always include appropriate positive controls such as HepG2, 293T, HeLa, or MCF-7 cell lysates, which are known to express detectable levels of PI4K2B .

How should results be interpreted when studying PI4K2B in cancer research contexts?

When analyzing PI4K2B expression data in cancer studies:

• Consider context-specific variations:

  • PI4K2B expression may vary significantly between different cancer types and stages

  • Loss of heterozygosity of the PI4K2B allele and PI4KIIβ underexpression have been associated with numerous cancers of epithelial origin

  • Compare results to appropriate normal tissue controls

• Correlate with functional phenotypes:

  • Assess whether PI4K2B expression levels correlate with invasion capacity

  • PI4K2B depletion has been shown to confer an aggressive invasive phenotype on minimally invasive HeLa and MCF-7 cell lines

  • Analyze matrix degradation capabilities in relation to PI4K2B expression

• Evaluate pathway interactions:

  • Consider PI4K2B's role in MT1-MMP trafficking and localization

  • Analyze colocalization with endosomal markers (Rab5, Rab7) and exocytic markers (Rab8)

  • Assess PI(4)P production and its downstream effects on signaling

• Account for technical considerations:

  • Standardize quantification methods across samples

  • Use multiple antibodies or detection methods to confirm findings

  • Include appropriate statistical analyses when comparing expression levels

These interpretive frameworks can help researchers accurately assess PI4K2B's role as a potential tumor suppressor and invasion regulator .

How can I distinguish between PI4K2B and other PI4K family members in my experiments?

Distinguishing between closely related PI4K family members requires careful experimental design:

• Antibody selection strategies:

  • Choose antibodies raised against unique regions of PI4K2B that show minimal homology with other family members

  • Verify specificity using knockdown or knockout controls for each isoform

  • Consider using antibodies that recognize post-translational modifications specific to PI4K2B

• Molecular weight differentiation:

  • PI4K2B has an observed molecular weight of 50-55 kDa

  • Compare to PI4KIIα (~55 kDa) and other family members with distinct molecular weights

  • Use high-resolution gels to separate closely migrating isoforms

• Localization-based differentiation:

  • PI4K2B is primarily cytosolic with some localization to endosomes, plasma membrane, and trans-Golgi network

  • PI4KIIα shows distinct localization patterns compared to PI4KIIβ

  • Use subcellular fractionation combined with immunoblotting to separate isoforms

• Functional discrimination:

  • Design experiments that target the unique functions of PI4K2B versus other isoforms

  • PI4KII isoforms synthesize separate pools of PI(4)P with distinct roles in TGN-to-endosome traffic

  • Selective depletion studies can help distinguish isoform-specific functions

These approaches help ensure that experimental findings can be correctly attributed to PI4K2B rather than related family members, which is crucial for accurate interpretation of results .

What emerging applications of PI4K2B antibodies show promise for translational research?

Several cutting-edge applications of PI4K2B antibodies hold potential for translational research:

• Biomarker development:

  • Given PI4K2B's role as a potential tumor suppressor, antibody-based assays could be developed to assess PI4K2B levels in patient samples

  • Correlation of PI4K2B expression with cancer progression could yield prognostic biomarkers

  • ELISA-based screening of patient serum/plasma could identify altered PI4K2B levels associated with disease states

• Therapeutic targeting validation:

  • Antibodies can confirm target engagement in drug development efforts

  • Monitor PI4K2B expression changes in response to experimental therapeutics

  • Validate the specificity of small molecule inhibitors being developed against PI4K family members

• Patient stratification approaches:

  • Immunohistochemical analysis of tumor biopsies using PI4K2B antibodies may help identify patient subgroups

  • Patients with low PI4K2B expression might benefit from specific therapeutic approaches targeting invasion mechanisms

  • Correlation with other molecular markers could improve precision medicine approaches

• Diagnostic imaging potential:

  • Development of labeled antibody fragments for molecular imaging

  • Potential applications in detecting tumors with altered PI4K2B expression

  • Research models using fluorescently labeled antibodies could translate to clinical diagnostics

These emerging applications highlight the importance of continued development and validation of high-quality PI4K2B antibodies for both research and potential clinical applications .

How might single-cell analysis techniques advance our understanding of PI4K2B function?

Single-cell technologies offer powerful approaches for elucidating PI4K2B biology:

• Single-cell immunoprofiling:

  • Use flow cytometry with PI4K2B antibodies to quantify expression heterogeneity within populations

  • Combine with markers of cell cycle, differentiation, or activation states

  • Assess correlations between PI4K2B levels and cellular phenotypes at single-cell resolution

• Spatial transcriptomics integration:

  • Combine PI4K2B immunostaining with spatial transcriptomics

  • Map PI4K2B protein expression to transcriptional profiles in tissue contexts

  • Identify neighborhood effects and microenvironmental influences on PI4K2B function

• Advanced imaging approaches:

  • Super-resolution microscopy with PI4K2B antibodies to precisely map subcellular localization

  • Live-cell imaging to track dynamic changes in PI4K2B distribution

  • Correlative light and electron microscopy to connect PI4K2B localization with ultrastructural features

• Single-cell proteomics:

  • Mass cytometry (CyTOF) incorporating PI4K2B antibodies

  • Simultaneous detection of PI4K2B with dozens of other proteins

  • Identification of previously unknown correlations with signaling pathways

These single-cell approaches could reveal how heterogeneous PI4K2B expression and function contribute to cellular behaviors, particularly in complex contexts like cancer progression and cellular differentiation .