CLINT1 Antibody

What is CLINT1 and what experimental applications are suitable for CLINT1 antibodies?

CLINT1 (also known as enthoprotin or epsin-4) is a 68-70 kDa protein belonging to the epsin family of endocytic adapter proteins. It interacts with clathrin, the adapter protein AP-1, and phosphoinositides, playing a role in trafficking between the trans-Golgi network and endosomes .

Validated applications for CLINT1 antibodies include:

For optimal results, it's recommended to titrate antibodies in each testing system as sample dependencies may affect performance .

How should CLINT1 antibodies be validated for specificity?

Antibody validation is crucial for ensuring research reproducibility. For CLINT1 antibodies, employ the following validation strategies:

• Orthogonal validation: Compare antibody-based detection with antibody-independent methods like mass spectrometry .

• Genetic knockout/knockdown validation:

  • Test antibodies on CLINT1 knockout or knockdown cells alongside wild-type controls.

  • The decreased signal in CLINT1-depleted samples validates specificity .

  • Research shows P/U ratios of endogenous CLINT1 were reduced by 2- and 20-fold in BIKE-depleted and BIKE-knockout cells respectively .

• Independent antibody validation: Use multiple antibodies targeting different epitopes of CLINT1 to confirm results .

• Recombinant expression validation: Test antibodies against cells overexpressing CLINT1 .

• Capture mass spectrometry: Confirm that immunoprecipitated proteins include CLINT1 .

A recent study demonstrated that implementing these five validation principles can significantly enhance antibody specificity assessment for research applications .

What is the expected molecular weight pattern for CLINT1 in Western blot applications?

When performing Western blot with CLINT1 antibodies, researchers should expect:

• Calculated molecular weight: 68 kDa

• Observed molecular weight: Multiple bands may appear:

  • Primary bands at 68 kDa (isoforms 1 and 2)

  • Additional band at 70-71 kDa (isoform 3)

  • Lower molecular weight bands may appear representing different phosphorylation states

The detection of multiple bands doesn't necessarily indicate non-specificity, as CLINT1 exists in multiple isoforms and phosphorylation states. When CLINT1-FLAG is pulled down and blotted with anti-FLAG antibody, an additional lower-molecular-weight band is often observed, potentially representing complexes formed between CLINT1-FLAG, endogenous BIKE, and endogenous CLINT1, or CLINT1 oligomers .

What are recommended sample preparation methods for optimal CLINT1 detection?

For robust and reproducible CLINT1 detection, follow these sample preparation guidelines:

For Western blot:

• Extract proteins using standard cell lysis buffers containing protease inhibitors

• Include phosphatase inhibitors if studying phosphorylated forms of CLINT1

• Load 20-50 μg of total protein lysate per lane

• Sample types with confirmed expression: HepG2, HEK-293, HeLa, Jurkat cells, and mouse brain tissue

For immunohistochemistry:

• Formalin-fixed, paraffin-embedded tissues are suitable

• Heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) is recommended

• Alternative: antigen retrieval with citrate buffer pH 6.0

• Human breast cancer tissue serves as a positive control

For immunofluorescence:

• Fixation with -20°C ethanol has been validated for HepG2 cells

• Alternative: 4% paraformaldehyde fixation followed by permeabilization

For immunoprecipitation:

• Use non-denaturing cell lysates for intracellular proteins

• For detecting CLINT1 interactions, cell media can be used for secreted proteins

How should storage and handling of CLINT1 antibodies be managed for long-term stability?

To maintain antibody integrity and performance over time:

• Store antibodies at -20°C as received

• For long-term storage, aliquot to avoid repeated freeze/thaw cycles

• Most CLINT1 antibodies are formulated in PBS (pH 7.3) containing 0.02% sodium azide and 50% glycerol

• Working dilutions should be prepared fresh before use

• Note that some commercially available antibodies (20μl sizes) contain 0.1% BSA

• Most CLINT1 antibodies are stable for one year after shipment when stored properly

Antibody aliquoting is generally unnecessary for -20°C storage of CLINT1 antibodies from certain manufacturers, but this may vary by supplier .

What methodologies can detect different phosphorylation states of CLINT1?

CLINT1 phosphorylation, particularly by BIKE at threonine 294 (T294), plays a crucial role in its binding to viral proteins and subsequent viral assembly processes . To study these phosphorylation events:

• Phosphorylation-specific antibodies:

  • While not described in the search results, phospho-specific antibodies targeting T294 would be ideal for direct detection.

  • In the absence of such antibodies, use general phospho-threonine antibodies after CLINT1 immunoprecipitation.

• Phos-tag gel electrophoresis:

  • Employ Phos-tag acrylamide gels which can separate phosphorylated and non-phosphorylated forms of CLINT1.

  • Research shows that upon longer exposure of Western blot membranes, lower-molecular-weight bands representing additional phosphorylation states of endogenous CLINT1 become visible .

• In vitro kinase assays:

  • Recombinant CLINT1 can be incubated with purified kinases (BIKE, AAK1, GAK) in the presence of [γ-32P]ATP.

  • Studies demonstrate that BIKE specifically phosphorylates the C-terminal domain of CLINT1, with high-intensity bands appearing that are absent in no-substrate controls .

• Phosphorylation site mutation:

  • Generate T294A mutants to prevent phosphorylation at this site.

  • Compare wild-type and mutant CLINT1 function in cellular assays.

• Phosphorylation quantification:

  • Use the P/U ratio (phosphorylated/unphosphorylated) methodology to quantify phosphorylation levels.

  • Research has shown P/U ratios of endogenous CLINT1 reduced by 2-fold in BIKE-depleted cells and 20-fold in BIKE-knockout cells .

What techniques are most effective for studying CLINT1's role in intracellular trafficking?

CLINT1 participates in clathrin-mediated budding from internal compartments and trafficking between the trans-Golgi network and endosomes . To investigate these functions:

• Live-cell imaging with fluorescently tagged CLINT1:

  • Generate cells expressing CLINT1-GFP/RFP fusion proteins.

  • Track vesicle movement in real-time.

  • Research has demonstrated that CLINT1 co-traffics with dengue virus particles .

• Co-localization studies:

  • Use dual-immunofluorescence with markers for different cellular compartments.

  • Markers include clathrin, AP-1, TGN46 (trans-Golgi), EEA1 (early endosomes).

  • Calculate Pearson's correlation coefficient to quantify co-localization.

• Proximity ligation assay (PLA):

  • Detect and visualize protein-protein interactions between CLINT1 and trafficking machinery components.

  • Particularly useful for transient interactions that may be difficult to capture by co-immunoprecipitation.

• Vesicle isolation and proteomics:

  • Isolate clathrin-coated vesicles through subcellular fractionation.

  • Analyze protein composition by mass spectrometry.

  • Compare vesicle content in CLINT1-depleted vs. control cells.

• Cargo trafficking assays:

  • Monitor transport of specific cargo proteins dependent on CLINT1.

  • Pulse-chase experiments with cargo proteins can reveal trafficking kinetics and defects.

• Electron microscopy:

  • Immunogold labeling of CLINT1 for ultrastructural localization.

  • Assess morphological changes in vesicular compartments upon CLINT1 depletion.

How can researchers effectively investigate the interaction between CLINT1 and viral proteins?

Studies have revealed that CLINT1 interacts with dengue virus (DENV) nonstructural 3 (NS3) protein, promoting viral assembly and egress . To further study CLINT1-viral protein interactions:

• Co-immunoprecipitation (co-IP) assays:

  • Transfect cells with constructs expressing tagged CLINT1 (e.g., CLINT1-FLAG).

  • Perform pulldown with anti-FLAG affinity gel followed by Western blot analysis.

  • Research has confirmed effective pulldown of FLAG-tagged proteins and co-immunoprecipitation of CLINT1 with interaction partners .

• Barcode fusion genetics-yeast two-hybrid (BFG-Y2H) screening:

  • For discovering novel CLINT1-viral protein interactions.

  • Research identified CLINT1 as a top BIKE interactor using this method .

• NanoLuciferase complementation assay:

  • Provides high-fidelity measurement of transient and weak interactions.

  • Can evaluate apparent affinities (Kds in the μM range) within appropriate subcellular compartments .

  • Normalize interaction signals to generate a normalized luminescence ratio (NLR).

• Immunofluorescence co-localization:

  • Study co-localization of CLINT1 with viral proteins during infection.

  • Live-cell imaging reveals CLINT1 co-trafficking with DENV particles .

• CRISPR/Cas9 knockout studies:

  • Generate CLINT1-knockout cell lines to assess effects on viral replication.

  • Complement with rescue experiments using wild-type or mutant CLINT1.

• Domain mapping:

  • Generate truncation mutants to identify which CLINT1 domains are required for viral protein binding.

  • Research shows that BIKE specifically phosphorylates the C-terminal domain of CLINT1 .

How can researchers distinguish between different CLINT1 isoforms?

CLINT1 exists in multiple isoforms that may have distinct functional roles. To differentiate between these isoforms:

• Western blot analysis with specific antibodies:

  • The anti-CLINT1 antibody detects three isoforms: two predominant isoforms (1 and 2) at 68 kDa and a lower intensity band corresponding to isoform 3 (70 kDa) .

  • Select antibodies recognizing epitopes specific to certain isoforms.

• RT-PCR for mRNA splice variants:

  • Design primers to detect levels and alternative splicing of transcripts.

  • For CLINT1 detection, primers used in research include: 5′-AAAGTGCGGGAGCTGGTTGAT-3′ and 5′-GCCGTTCTCGTCGACACAATGAT-3′ .

• Overexpression of tagged isoforms:

  • Generate constructs expressing individual isoforms with distinguishable tags.

  • The CLINT1-FLAG plasmid encoding isoform 1 enhances the signal of the top band (~71 kDa) .

• Mass spectrometry analysis:

  • Identify unique peptides corresponding to specific isoforms.

  • Quantify relative abundance of each isoform.

• Isoform-specific knockdown:

  • Design siRNAs targeting unique regions of specific isoforms.

  • Analyze the functional impact of selectively reducing individual isoforms.

• Immunofluorescence with isoform-specific antibodies:

  • Examine potential differences in subcellular localization between isoforms.

What methodologies are most appropriate for studying CLINT1's role in developmental processes?

Research indicates CLINT1 plays a role in epidermal development, with its deficiency leading to persistent inflammation in epidermal tissues . To investigate CLINT1's developmental functions:

• Animal model studies:

  • Use zebrafish CLINT1 mutants which display enhanced proliferation, cell death, and leukocyte infiltration as early as 36 hpf .

  • Quantify neutrophil displacement outside the caudal hematopoietic tissue (CHT) .

• Immunostaining for inflammatory markers:

  • Co-immunolabeling with antibodies to Mpo and L-plastin (Lcp1), a general leukocyte marker .

  • Identify L-plastin-positive/Mpo-positive (neutrophils) and L-plastin-positive/Mpo-negative (macrophages and other leukocytes) cell populations.

• Gene expression analysis:

  • Analyze inflammatory cytokine expression (e.g., IL1β) in CLINT1-deficient models .

  • Use RT-PCR with primers such as:

    • IL1β: 5′-GCATGCGGGCAATATGAAGT-3′ and 5′-GTTCACTTCACGCTCTTGGATG-3′ .

• Cell proliferation and death assays:

  • Examine cell proliferation and death in CLINT1 mutants at different developmental stages.

  • Significant elevations in epidermal proliferation and cell death can be observed in CLINT1 mutants .

• Tissue-specific CLINT1 knockdown/knockout:

  • Generate conditional knockout models to assess tissue-specific functions.

  • Perform rescue experiments to confirm phenotype specificity.

• Time-course analyses:

  • Study developmental processes at multiple time points to establish causality.

  • Research shows significant elevations in epidermal proliferation and cell death in CLINT1 mutants as early as 36 hpf, with leukocyte infiltration observed at 48 hpf .

How should researchers troubleshoot inconsistent results with CLINT1 antibodies?

Inconsistent antibody performance is a common challenge in research. For CLINT1 antibodies specifically:

• Validate antibody specificity:

  • Test on CLINT1 knockout cells alongside controls.

  • Research indicates many laboratory experiments are unreliable due to inconsistent immunohistochemical staining caused by improper antibody validation .

  • It's estimated that at minimum, half of manuscripts contain potentially incorrect immunohistochemical staining results due to lack of best practice antibody validation .

• Optimize antibody concentration:

  • Titrate antibodies to determine optimal working concentration.

  • Follow recommended dilutions (e.g., 1:1000-1:4000 for WB, 1:50-1:500 for IHC) .

• Modify sample preparation:

  • Test different antigen retrieval methods for IHC:

    • Tris-EDTA buffer (pH 9.0) (primary recommendation)

    • Citrate buffer (pH 6.0) (alternative method)

  • For Western blot, try different lysis buffers and ensure adequate denaturation.

• Control for phosphorylation status:

  • Include phosphatase inhibitors if studying phosphorylated forms.

  • CLINT1 phosphorylation is regulated by kinases like BIKE .

• Compare multiple antibodies:

  • Use antibodies targeting different epitopes of CLINT1.

  • Available antibodies target different regions:

    • AA 161-261

    • AA 1-625

    • AA 222-249

    • AA 229-278

    • AA 108-157

    • AA 250-400

• Assess antibody cross-reactivity:

  • Test on multiple cell types to ensure consistent results.

  • Consider species cross-reactivity when working with non-human models.

A systematic approach to troubleshooting, combined with proper validation, will help ensure reliable and reproducible results with CLINT1 antibodies.