PLEKHF2 Antibody

What is PLEKHF2 and why is it important in cellular research?

PLEKHF2 (Phafin2) belongs to the Phafin protein family, which contains both Pleckstrin Homology (PH) and FYVE domains. These membrane-associated proteins serve as important "membrane readers" that facilitate signaling and trafficking pathways in eukaryotic cells . The protein's dual-domain structure allows it to interact with various protein and lipid ligands, positioning it as a potential intracellular adaptor or targeting molecule in cellular processes. Understanding PLEKHF2 is particularly relevant in investigating cellular signaling pathways and potential disease mechanisms, as Phafin proteins have been implicated in conditions such as idiopathic pulmonary fibrosis and cancer .

What types of PLEKHF2 antibodies are available for research applications?

Multiple PLEKHF2 antibody variants are available, each with specific characteristics suitable for different experimental designs:

Researchers should select antibodies based on their experimental questions, target species, and planned applications. For instance, studies requiring cross-species reactivity would benefit from the N-terminal antibody, while human-specific investigations might utilize the central region antibody .

How should I determine the optimal working dilution for PLEKHF2 antibodies in different applications?

Determining the optimal working dilution for PLEKHF2 antibodies requires systematic titration based on your specific experimental conditions. While manufacturers provide recommended dilution ranges (e.g., 1:100-1:500 for Western blot applications ), these serve only as starting points. The optimal dilution must balance obtaining strong specific signals while minimizing background.

A methodological approach includes:

• Prepare a dilution series spanning the recommended range (e.g., 1:100, 1:200, 1:500, 1:1000, 1:2000)

• Perform your application (WB, IHC, ELISA) using identical samples across all dilutions

• Evaluate signal-to-noise ratio at each dilution

• Select the highest dilution that maintains strong specific signals with minimal background

For PLEKHF2 antibodies targeting the N-terminus, manufacturers specifically note that "optimal working dilution should be determined by the investigator" , emphasizing the importance of this optimization step for each unique experimental system.

What positive controls are recommended for validating PLEKHF2 antibody specificity?

Validating antibody specificity is essential for ensuring experimental rigor. For PLEKHF2 antibodies, positive controls should be selected based on known expression patterns of the protein. While specific PLEKHF2 expression data is limited in the provided search results, the approach should follow standard validation principles:

• Use tissues/cells with confirmed PLEKHF2 expression

• Consider knockout/knockdown models as negative controls

• Test multiple antibodies targeting different epitopes to confirm specificity

Based on sequence homology analysis reported for the N-terminal PLEKHF2 antibody, multiple species show high conservation: "Human, Gorilla, Gibbon, Monkey, Galago, Marmoset, Mouse, Rat, Elephant, Rabbit, Opossum, Zebra finch, Chicken, Platypus, Lizard (100%), Panda, Dog, Bovine, Bat, Horse, Guinea pig (92%), Drosophila (84%)" . This suggests tissues from these organisms could potentially serve as positive controls, with appropriate species-matched secondary antibodies.

What are the key considerations when selecting between different PLEKHF2 antibody formats (conjugated vs. unconjugated)?

The selection between conjugated and unconjugated PLEKHF2 antibodies depends on your experimental design, detection system, and workflow considerations:

Most PLEKHF2 antibodies in the search results are offered in unconjugated format , though APC-conjugated variants are available for specific applications . When using unconjugated antibodies, researchers must account for additional protocol steps, including secondary antibody incubation and appropriate blocking. Conversely, conjugated antibodies offer streamlined workflows but less flexibility in detection methods. Your choice should be guided by the specific requirements of your experimental system and available detection infrastructure.

How can I optimize PLEKHF2 antibody performance in challenging applications like co-immunoprecipitation or ChIP?

Optimizing PLEKHF2 antibody performance for advanced applications requires attention to antibody quality, buffer conditions, and protocol modifications:

For co-immunoprecipitation (Co-IP):

• Use immunoaffinity-purified antibodies like those targeting the N-terminus of PLEKHF2

• Pre-clear lysates to reduce non-specific binding

• Optimize antibody concentration (typically higher than for Western blot)

• Consider cross-linking the antibody to beads to prevent heavy chain interference

• Use gentle lysis conditions to preserve protein-protein interactions

For chromatin immunoprecipitation (ChIP), although not explicitly mentioned in search results for PLEKHF2, general principles apply:

• Confirm antibody specificity via Western blot first

• Test different fixation times to optimize chromatin shearing

• Include appropriate controls (input, IgG, positive locus)

• Perform antibody titration specific to ChIP conditions

What approaches can resolve conflicting results when using different PLEKHF2 antibodies?

When different PLEKHF2 antibodies yield conflicting results, a systematic troubleshooting approach is essential:

• Epitope mapping analysis: Compare the binding regions of the antibodies (e.g., N-terminal versus central region AA 71-98 ). Different domains may be accessible in various experimental conditions or differentially modified in certain cellular states.

• Protocol standardization: Standardize all variables except the antibody:

  • Sample preparation method

  • Blocking conditions

  • Incubation times and temperatures

  • Detection systems

• Validation with orthogonal methods: Confirm findings using:

  • mRNA expression analysis

  • Overexpression studies

  • Knockout/knockdown controls

  • Mass spectrometry

• Antibody validation: Perform cross-validation experiments:

  • Pre-absorption with immunizing peptide

  • Western blot to confirm specificity

  • Testing in cell lines with known expression levels

The resolution may reveal that different antibodies detect distinct conformations, splice variants, or post-translational modifications of PLEKHF2, providing complementary rather than conflicting information.

How does the dual domain structure of PLEKHF2 (PH and FYVE domains) impact antibody selection for specific signaling pathway analyses?

The dual domain architecture of PLEKHF2, containing both PH and FYVE domains, creates important considerations for antibody selection when investigating specific signaling pathways:

PLEKHF2's PH and FYVE domains mediate distinct interactions: PH domains typically bind phosphoinositides and proteins, while FYVE domains specifically recognize phosphatidylinositol 3-phosphate (PI3P) in cellular membranes . This domain architecture suggests PLEKHF2 may localize to specific membrane compartments and participate in multiple signaling pathways.

For targeted signaling pathway analysis:

• Domain-specific antibodies: Select antibodies targeting the relevant domain:

  • N-terminal antibodies may detect the PH domain region

  • Central region antibodies (AA 71-98) might reveal different interaction patterns

• Functional considerations:

  • For endosomal trafficking studies, FYVE domain accessibility is critical

  • For cytosolic signaling investigations, PH domain recognition may be more relevant

• Experimental design implications:

  • Membrane fractionation studies require antibodies that recognize native conformations

  • Studies of protein-protein interactions might need antibodies that don't interfere with binding surfaces

• Combined approaches: For comprehensive analysis, use multiple antibodies recognizing different epitopes to distinguish domain-specific functions and interactions.

Understanding the specific signaling context of your research question should guide antibody selection to ensure the relevant functional domain or protein interaction surface is appropriately detected.

What is known about PLEKHF2 antibody applications in cancer research?

The involvement of Phafin family proteins, including PLEKHF2, in cancer research is an emerging area of investigation. While specific cancer-related applications of PLEKHF2 antibodies are not extensively detailed in the provided search results, the fundamental understanding of Phafin proteins provides context for potential research directions:

A recent review indicates that Phafin proteins (including PLEKHF2/Phafin2) have implications in cancer research . As membrane-associated proteins that mediate signaling and trafficking pathways, they may influence critical cellular processes relevant to tumorigenesis and progression.

For cancer research applications, PLEKHF2 antibodies could be utilized in:

• Expression profiling: Analyzing PLEKHF2 expression across cancer types and stages using immunohistochemistry (IHC) applications supported by available antibodies

• Signaling pathway investigation: Exploring PLEKHF2's role in cancer-related signaling pathways through co-immunoprecipitation and Western blot approaches

• Subcellular localization studies: Determining if PLEKHF2 localization changes in malignant versus normal tissues using immunofluorescence techniques

• Biomarker evaluation: Assessing PLEKHF2 as a potential diagnostic or prognostic biomarker through quantitative approaches like ELISA, for which specific antibodies are available

Researchers investigating PLEKHF2 in cancer contexts should first validate antibody performance in relevant cancer cell lines or tissues before proceeding to larger studies.

How can PLEKHF2 antibodies be utilized in studying inflammatory and fibrotic diseases?

Based on emerging research on Phafin proteins, PLEKHF2 antibodies may have valuable applications in studying inflammatory and fibrotic diseases:

The search results indicate that Phafin1, a related protein in the same family as PLEKHF2 (Phafin2), shows significantly increased levels in the lungs of idiopathic pulmonary fibrosis patients compared to normal subjects . This suggests a potential role for Phafin proteins in the pathogenesis of fibrotic conditions and provides a foundation for investigating PLEKHF2 in similar disease contexts.

Methodological approaches for studying PLEKHF2 in inflammatory and fibrotic diseases include:

• Tissue expression analysis: Using immunohistochemistry-validated PLEKHF2 antibodies to compare expression patterns in normal versus diseased tissues

• Cell-type specific studies: Employing immunofluorescence to identify which cell populations alter PLEKHF2 expression during disease progression

• Signaling pathway investigation: Using Western blotting and co-immunoprecipitation to determine how PLEKHF2 interactions change in inflammatory conditions

• Intervention response assessment: Measuring PLEKHF2 expression changes following treatment with anti-inflammatory or anti-fibrotic agents

When designing such studies, researchers should consider using multiple PLEKHF2 antibodies targeting different epitopes to ensure comprehensive detection and validate findings across methodologies.

What strategies can address non-specific binding when using PLEKHF2 antibodies in Western blotting?

Non-specific binding in Western blotting with PLEKHF2 antibodies can be systematically addressed through multiple optimization strategies:

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Include blocking agent in antibody dilution buffer

• Antibody dilution adjustment:

  • Titrate antibody concentration beyond manufacturer recommendations

  • For PLEKHF2 antibodies, starting dilutions range from 1:100-1:500 for Western blotting

  • Consider more dilute solutions (1:1000-1:20000) for cleaner results

• Buffer modifications:

  • Add 0.1-0.5% detergent (Tween-20) to washing and antibody dilution buffers

  • Increase salt concentration (150-500 mM NaCl) to reduce ionic interactions

  • Consider adding 5% serum from the secondary antibody host species

• Sample preparation refinement:

  • Include protease inhibitors to prevent degradation products

  • Optimize protein loading (10-30 μg typically)

  • Ensure complete denaturation of samples

• Membrane handling:

  • Cut membranes to minimize antibody usage

  • Consider PVDF versus nitrocellulose based on background issues

  • Ensure adequate washing between steps (at least 3 x 10 minutes)

For the PLEKHF2 N-terminal antibody, immunoaffinity purification should reduce non-specific binding, but additional optimization may still be necessary for challenging samples.

How can I preserve PLEKHF2 antibody activity during long-term storage and repeated use?

Maintaining PLEKHF2 antibody activity over time requires careful attention to storage and handling procedures:

• Optimal storage conditions:

  • Store at -20°C for long-term preservation (up to 1 year)

  • For short-term use (less than 1 week), 4°C storage is acceptable

  • Add stabilizing proteins (BSA 0.1-1%) if not already present in formulation

• Aliquoting strategy:

  • Divide antibody into small single-use aliquots immediately upon receipt

  • Use small (10-20 μL) aliquots to minimize freeze-thaw cycles

  • Label aliquots with dilution factors and dates

• Preservation additives:

  • Commercial antibody stabilizers may be added if not pre-formulated

  • Glycerol (final concentration 30-50%) prevents freezing at -20°C and reduces freeze-thaw damage

  • Sodium azide (0.02-0.09%) prevents microbial growth

• Handling practices:

  • Allow antibodies to equilibrate to room temperature before opening vials

  • Centrifuge briefly before opening to collect solution at the bottom

  • Use sterile technique when accessing antibody solutions

• Reconstitution of lyophilized antibodies:

  • Follow manufacturer's specific instructions

  • For PLEKHF2 antibodies, distilled water is typically recommended

  • Reconstitute only the amount needed and aliquot remaining lyophilized material

Manufacturers recommend avoiding repeated freeze-thaw cycles for PLEKHF2 antibodies , underscoring the importance of proper aliquoting immediately upon receipt.