pdik1l Antibody

What is PDIK1L and why is it important in research?

PDIK1L (PDLIM1 Interacting Kinase 1 Like) is a serine/threonine protein kinase belonging to the protein kinase superfamily. It has gained research interest due to its role in cellular signaling pathways and potential connections to various pathological conditions. Studies have shown that PDIK1L forms part of a dual phospho-catalytic signaling complex with SCP4 and STK35 . The protein has been particularly studied as a biomarker for endometriosis, with research indicating that anti-PDIK1L autoantibodies may serve as potential diagnostic markers for this condition .

What is the subcellular localization of PDIK1L protein?

PDIK1L is primarily localized in the nucleus, as demonstrated by fluorescence microscopy studies using EGFP-tagged constructs. Research indicates that the N-terminus of the protein contains functional nuclear and nucleolar localization signals. When the N-terminal 196 amino acids are deleted, the protein distributes diffusely throughout both the cytoplasm and nucleus, confirming the importance of this region for proper subcellular targeting . Western blot analyses of nuclear lysates from various cell types, including HEK, HeLa, and endothelial cells, have verified this nuclear localization pattern .

What types of PDIK1L antibodies are available for research applications?

Several types of PDIK1L antibodies are available for research, including:

These antibodies have been validated for various applications, with most showing reactivity against human PDIK1L and some cross-reacting with mouse PDIK1L .

How should I validate a PDIK1L antibody for my specific research application?

Validation of PDIK1L antibodies should follow a multi-step approach:

• Western blot analysis: Verify specificity by detecting the expected band size (~39 kDa) in appropriate cell or tissue lysates . Compare with positive controls expressing PDIK1L.

• Antibody titration: Perform dilution series (typically 1/500 - 1/2000 for WB, 1/100 - 1/300 for IHC, and 1/40000 for ELISA) to determine optimal antibody concentration for your specific sample type and detection method .

• Cross-reactivity assessment: If working with non-human samples, verify cross-reactivity with your species of interest as not all anti-PDIK1L antibodies recognize orthologous proteins across species .

• Knockout/knockdown controls: When possible, include PDIK1L knockout or knockdown samples to confirm specificity and rule out non-specific binding.

• Subcellular localization confirmation: For immunofluorescence applications, verify that staining patterns match expected nuclear localization of PDIK1L .

What are the recommended protocols for detecting PDIK1L using Western blotting?

For optimal Western blot detection of PDIK1L:

• Sample preparation: Prepare whole cell lysates or nuclear extracts (preferred due to PDIK1L's nuclear localization) in Laemmli buffer.

• Gel electrophoresis: Separate proteins using 10-12% SDS-PAGE.

• Transfer conditions: Transfer to nitrocellulose membrane at 200 mAmp for 60 min at 4°C .

• Blocking: Block membranes with 5% (w/v) nonfat milk in TBST.

• Primary antibody incubation: Use anti-PDIK1L antibody at dilutions of 1:500-1:2000, incubate overnight at 4°C.

• Secondary antibody: Apply HRP-conjugated secondary antibody at 1:5000 dilution.

• Detection: Develop using chemiluminescent substrate.

• Expected result: PDIK1L appears as a band at approximately 39 kDa .

How can I optimize immunohistochemistry (IHC) protocols for PDIK1L detection in tissue samples?

For effective IHC detection of PDIK1L in tissue samples:

• Tissue preparation: Use formalin-fixed, paraffin-embedded sections (4-6 μm thickness).

• Antigen retrieval: Perform heat-induced epitope retrieval in citrate buffer (pH 6.0).

• Blocking: Block endogenous peroxidase activity with 3% H₂O₂, followed by protein blocking with 5% normal serum.

• Antibody dilution: Use antibody at 1:100-1:300 dilution for optimal results .

• Incubation conditions: Incubate with primary antibody overnight at 4°C.

• Detection system: Use biotin-streptavidin or polymer-based detection systems.

• Counterstaining: Counterstain with hematoxylin.

• Controls: Include negative controls (antibody diluent only) and positive controls (tissues known to express PDIK1L, such as breast carcinoma tissue) .

• Expected localization: Nuclear staining pattern.

How can PDIK1L antibodies be used to study the SCP4-STK35/PDIK1L complex?

The SCP4-STK35/PDIK1L complex represents a dual phospho-catalytic signaling system . To study this complex:

• Co-immunoprecipitation (Co-IP): Use anti-PDIK1L antibodies to immunoprecipitate the protein complex from cell lysates, followed by Western blotting for STK35 and SCP4. Research has shown that HA-tagged STK35 and PDIK1L efficiently associate with SCP4 wild-type proteins .

• Proximity ligation assay (PLA): Apply this technique to visualize and quantify PDIK1L-SCP4-STK35 interactions in situ.

• Mutant analysis: Compare interactions between wild-type proteins and mutants (e.g., SCP4 D293A) to determine critical binding domains. Research indicates that SCP4 D293A mutation diminishes interaction with STK35 and PDIK1L .

• Mass spectrometry validation: Perform MS analysis of immunopurified HA-PDIK1L complexes to identify and quantify associated proteins. Previous research identified endogenous SCP4 as the most enriched protein in PDIK1L immunoprecipitates .

• Functional studies: Combine antibody-based detection methods with kinase activity assays to correlate protein interactions with phosphorylation status and downstream signaling effects.

What are the considerations for using PDIK1L antibodies in studying endometriosis biomarkers?

Anti-PDIK1L autoantibodies have been investigated as potential biomarkers for endometriosis:

• Patient cohort selection: Include appropriate case-control groups. Previous studies examined 69 patients with endometriosis, 38 disease control patients without endometriosis, and 44 healthy volunteers .

• Assay development: Establish ELISA methods using recombinant PDIK1L proteins as capture antigens to detect serum autoantibodies.

• Performance metrics: Analyze sensitivity, specificity, and accuracy. Research found that anti-PDIK1L autoantibody assays showed 59.4% sensitivity and 72.8% accuracy in endometriosis diagnosis, outperforming CA125 (36.2% and 62.9%, respectively) .

• Early detection potential: Evaluate ability to detect early-stage disease. Studies suggest anti-PDIK1L autoantibodies can detect endometriosis in early stages .

• Meta-analysis considerations: Current meta-analyses show heterogeneity in results. One meta-analysis found no significant relationship between anti-PDIK1L autoantibodies and endometriosis susceptibility (OR: 1.434, CI 95%: 0.241-8.528, p=0.692) .

• Regional differences: Consider geographical variations in autoantibody prevalence, as meta-analyses have identified region-specific heterogeneity in autoantibody studies .

How can I address non-specific binding issues when using PDIK1L antibodies?

Non-specific binding can compromise experimental results. To minimize this issue:

• Optimize blocking conditions: Test different blocking agents (BSA, milk, serum) and concentrations.

• Adjust antibody dilution: Further dilute primary antibody if background is high. Consider following specific recommendations: 1/500-1/2000 for WB, 1/100-1/300 for IHC, and 1/40000 for ELISA .

• Include appropriate controls: Use PDIK1L-negative tissues or cells as negative controls.

• Increase washing stringency: Extend wash time or add low concentrations of detergent to wash buffers.

• Pre-absorb antibody: For polyclonal antibodies, consider pre-absorption with tissues or cell lysates known to be negative for PDIK1L.

• Use monoclonal alternatives: If polyclonal antibodies show high background, consider switching to monoclonal antibodies with higher specificity .

• Affinity purification: Use antibodies purified by affinity chromatography using epitope-specific immunogens .

What are the major technical challenges in detecting PDIK1L protein in different experimental systems?

Researchers face several challenges when detecting PDIK1L:

• Low endogenous expression: PDIK1L may be expressed at low levels in certain tissues or cell types, requiring sensitive detection methods.

• Nuclear localization: The predominant nuclear localization of PDIK1L necessitates effective nuclear extraction protocols for biochemical analyses .

• Protein-protein interactions: PDIK1L's interactions with binding partners like SCP4 may mask epitopes and affect antibody recognition .

• Cross-reactivity concerns: Some antibodies may cross-react with related kinases, particularly STK35, which shares sequence homology with PDIK1L .

• Species-specific detection: Not all antibodies recognize PDIK1L across different species; verify cross-reactivity with your experimental model .

• Isoform specificity: Consider potential isoforms and ensure your antibody recognizes the relevant variant for your research question.

• Post-translational modifications: Phosphorylation or other modifications may affect epitope accessibility and antibody binding efficiency.

How are PDIK1L antibodies being utilized in emerging research on kinase signaling networks?

PDIK1L antibodies are increasingly valuable in studying complex kinase networks:

• Dual phospho-catalytic signaling: Research utilizing PDIK1L antibodies has revealed its role in the SCP4-STK35/PDIK1L complex, representing a novel dual phospho-catalytic signaling system with both kinase and phosphatase activities .

• Interactome mapping: Antibody-based affinity purification coupled with mass spectrometry is identifying novel PDIK1L interaction partners beyond established associations with SCP4 and STK35.

• Tissue-specific functions: Immunohistochemical analyses using PDIK1L antibodies are uncovering tissue-specific expression patterns that may indicate specialized functions in different cellular contexts.

• Disease associations: Beyond endometriosis, researchers are investigating PDIK1L's potential roles in other pathological conditions through antibody-based tissue profiling.

• Phosphoproteome analysis: Combining PDIK1L antibodies with phospho-specific detection methods is helping to identify downstream substrates and signaling pathways.

• Evolutionary conservation: Cross-species reactive antibodies are enabling comparative studies of PDIK1L function across evolutionary lineages .

What novel experimental approaches are being developed for studying PDIK1L function using antibody-based techniques?

Innovative approaches using PDIK1L antibodies include:

• Proximity-dependent biotinylation (BioID/TurboID): Coupling PDIK1L with promiscuous biotin ligases allows identification of proximal proteins in living cells, complementing traditional antibody-based co-IP methods.

• Single-cell analysis: Combining PDIK1L antibodies with single-cell technologies is revealing cell-to-cell variation in expression and localization patterns.

• Live-cell imaging: Development of cell-permeable antibody fragments or nanobodies against PDIK1L may enable real-time tracking of protein dynamics.

• CUT&RUN and CUT&Tag: Using PDIK1L antibodies in these chromatin profiling techniques can map potential genomic associations with higher resolution than traditional ChIP-seq.

• Antibody-drug conjugates for targeted inhibition: Coupling PDIK1L antibodies with kinase inhibitors or degraders for targeted delivery to specific cell populations.

• Multi-parameter imaging: Combining PDIK1L antibodies with other markers in multiplexed imaging approaches to understand spatial relationships in tissue contexts.

• 3D organoid systems: Applying PDIK1L antibodies to study kinase function in more physiologically relevant three-dimensional culture systems.