IFT140 Antibody

What is IFT140 and why is it important in research?

IFT140 is a critical subunit of the intraflagellar transport (IFT) complex A. It plays an essential role in the genesis, resorption, and signaling of primary cilia, which are microtubule-based sensory organelles present on the surface of most quiescent mammalian cells. These organelles receive environmental signals such as fluid flow, light, or odors, and transduce them to the nucleus. Research on IFT140 is particularly important because mutations in this gene are associated with ciliopathies, including skeletal and chondral development disorders. Studies have shown that loss of IFT140 in mouse models results in renal cystic disease, highlighting its significance in kidney development and function .

How do I choose the appropriate IFT140 antibody for my research?

Selecting the appropriate IFT140 antibody depends on your specific research applications and the species you are studying. Consider these factors:

• Target specificity: Ensure the antibody detects endogenous levels of total IFT140 in your species of interest.

• Host species: Most available IFT140 antibodies are rabbit polyclonal, which may influence your experimental design if using other antibodies concurrently.

• Reactivity: Verify that the antibody has been validated for your research organism (commonly human, mouse, or rat).

• Application compatibility: Different antibodies are optimized for specific techniques like Western blotting, immunohistochemistry, immunofluorescence, or immunoprecipitation.

• Target region: Consider whether you need an antibody targeting the N-terminal, C-terminal, or internal regions of IFT140 .

Commercial IFT140 antibodies are available with validated reactivity to human, mouse, and rat samples, with predicted cross-reactivity to other mammalian species including pig, bovine, horse, and sheep in some cases .

What validation should I perform before using an IFT140 antibody in my experiments?

Comprehensive validation is crucial before incorporating an IFT140 antibody into your research:

• Positive control testing: Confirm antibody performance using tissues/cells known to express IFT140, such as human skeletal muscle tissue or testis tissue from mouse or rat models .

• Western blot verification: Run a Western blot to confirm the antibody detects protein of the expected molecular weight (approximately 150-165 kDa) .

• Knockout/knockdown controls: If available, include IFT140 knockout or knockdown samples to verify specificity.

• Cross-reactivity assessment: Test for potential cross-reactivity with other IFT complex proteins, particularly if studying protein interactions.

• Concentration optimization: Determine optimal working dilutions for your specific application (e.g., WB: 1:1000-1:6000, IHC: 1:50-1:500) .

Begin with manufacturer-recommended dilutions and optimize based on your specific experimental conditions, sample types, and detection methods.

What are the optimal conditions for using IFT140 antibody in Western blotting?

For optimal Western blotting results with IFT140 antibody:

• Sample preparation:

  • For cell lines: HeLa and HepG2 cells have been successfully used with IFT140 antibodies

  • For tissue samples: Mouse and rat testis tissues provide reliable positive controls

• Dilution ranges:

  • Start with 1:1000 and optimize as needed (range typically 1:1000-1:6000)

  • Use 5% BSA in TBST for antibody dilution to minimize background

• Detection considerations:

  • Expected molecular weight: 150-165 kDa

  • Use appropriate secondary antibody (anti-rabbit IgG for most commercial options)

  • Consider enhanced chemiluminescence for detection with adequate exposure time

• Buffer conditions:

  • Transfer buffer optimization is critical for high molecular weight proteins like IFT140

  • Consider reduced methanol concentration and addition of SDS (0.1%) to transfer buffer to improve transfer efficiency

• Controls:

  • Include positive controls (HEK293 cells expressing IFT140)

  • Use appropriate loading controls based on your experimental design

How can I optimize immunoprecipitation protocols using IFT140 antibodies?

For successful immunoprecipitation of IFT140 and its interacting partners:

• Antibody selection:

  • Choose antibodies specifically validated for IP applications

  • Consider using 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate

• Tagging strategies:

  • N-terminal Strep/FLAG-tagged IFT140 constructs have proven effective for IP experiments

  • Consider co-transfection approaches for studying specific interactions (e.g., with TULP3)

• Lysis conditions:

  • Use gentle lysis buffers to preserve protein-protein interactions

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if studying phosphorylation status

• Elution and detection:

  • SDS-PAGE followed by Western blotting using specific antibodies for IFT140 and potential interacting partners

  • Mass spectrometry approaches have been used successfully to identify IFT140 interactome

• Controls:

  • Include negative controls (e.g., RAF1 has been used as a negative control in pull-down experiments)

  • Use IgG controls to identify non-specific binding

What are the key considerations for immunofluorescence studies with IFT140 antibodies?

Optimize immunofluorescence experiments with IFT140 antibodies by considering:

• Fixation and permeabilization:

  • Paraformaldehyde (4%) fixation followed by Triton X-100 (0.1-0.3%) permeabilization works well for most applications

  • Duration of fixation may need optimization depending on tissue/cell type

• Antibody dilution:

  • For IF/ICC: Use dilutions between 1:400-1:1600

  • For IF-P (tissue sections): Use dilutions between 1:200-1:800

• Blocking conditions:

  • 5-10% normal serum (matching the species of secondary antibody) in PBS with 0.1% Triton X-100

  • Consider adding 1-3% BSA to reduce background

• Co-staining considerations:

  • IFT140 is typically visualized in cilia structures

  • Consider co-staining with other ciliary markers (acetylated tubulin, gamma-tubulin) to provide context

  • Plan antibody combinations carefully to avoid host species conflicts

• Microscopy settings:

  • High-resolution confocal microscopy is often necessary to visualize ciliary structures

  • Z-stack imaging may be required to fully capture ciliary morphology

What are common troubleshooting issues with IFT140 antibodies in Western blotting?

When experiencing difficulties with IFT140 antibody performance in Western blotting:

• No signal or weak signal:

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

  • Extend primary antibody incubation time (overnight at 4°C)

  • Check protein loading (minimum 30-50 μg total protein recommended)

  • Verify transfer efficiency, especially for this high molecular weight protein (165 kDa)

  • Consider adding SDS (0.1%) to transfer buffer to improve transfer of large proteins

• Multiple bands or unexpected band size:

  • Verify antibody specificity using knockout/knockdown controls

  • Check for protein degradation by adding more protease inhibitors

  • Look for potential post-translational modifications that could alter migration

  • Confirm gel percentage is appropriate for resolving high molecular weight proteins (6-8% recommended)

• High background:

  • Increase blocking time and concentration

  • Wash more extensively between antibody incubations

  • Dilute primary antibody further

  • Use highly purified antibody preparations (e.g., antigen affinity purified)

• Sample-dependent issues:

  • Different sample types may require optimization (cell lines vs. tissue lysates)

  • Test antibody performance in known positive samples (HeLa cells, mouse testis)

How can I distinguish between specific and non-specific binding in immunohistochemistry?

To ensure specific staining in immunohistochemistry with IFT140 antibodies:

• Antigen retrieval optimization:

  • Different antibodies may require specific antigen retrieval methods

  • Try both citrate buffer (pH 6.0) and TE buffer (pH 9.0) to determine optimal conditions

• Antibody titration:

  • Perform dilution series (starting from 1:50-1:500) to determine optimal concentration

  • Excessive antibody concentration often increases background staining

• Controls for validation:

  • Include tissues known to express IFT140 (mouse testis tissue, human skeletal muscle)

  • Include negative control tissues (low or no expression)

  • Use sections processed identically but without primary antibody

  • When possible, include IFT140 knockout/knockdown tissues

• Blocking optimization:

  • Test different blocking reagents (BSA, normal serum, commercial blockers)

  • Extend blocking time if non-specific binding persists

• Pattern analysis:

  • Specific staining should correspond to expected cellular localization (primary cilia)

  • Compare staining patterns with published literature

How can I use IFT140 antibodies to study protein-protein interactions in the IFT-A complex?

To investigate protein-protein interactions involving IFT140:

• Co-immunoprecipitation approaches:

  • Use IFT140 antibodies to pull down the protein and its interacting partners

  • Western blot with antibodies against suspected interacting proteins

  • Alternatively, use N-terminal tagged (Strep/FLAG) IFT140 constructs for pull-down experiments

• Mass spectrometry analysis:

  • Perform affinity purification followed by LC-MS/MS analysis

  • Use label-free quantification to identify and quantify interaction partners

  • Statistical analysis (e.g., Student's t-test p-value ≤ 0.05) to determine significant interactions

• Validation strategies:

  • Confirm interactions using reciprocal co-IP experiments

  • Use different tags and antibodies to verify results

  • Consider proximity ligation assays for in situ confirmation

• Known interactors to investigate:

  • All components of the IFT-A complex

  • TULP3, which has been confirmed as an interactor of IFT140

  • Additional novel interactors identified in recent studies

• Controls:

  • Use unrelated proteins (e.g., RAF1) as negative controls

  • Include IgG controls to identify non-specific binding

How can IFT140 antibodies be used to study ciliopathy mechanisms?

For investigating mechanisms underlying ciliopathies associated with IFT140 dysfunction:

• Mutation effect analysis:

  • Compare IFT140 expression and localization in wildtype vs. mutant cells/tissues

  • Assess stability of the IFT-A complex in disease-relevant mutations

  • Analyze specific protein-protein interactions disrupted by pathogenic mutations

• Functional studies:

  • Examine ciliogenesis in cells with IFT140 mutations using immunofluorescence

  • Assess ciliary transport using live imaging with IFT140 antibodies or tagged constructs

  • Evaluate downstream signaling pathways affected by IFT140 dysfunction

• Disease models:

  • Use IFT140 antibodies to characterize mouse models of ciliopathies

  • Study renal phenotypes, as loss of IFT140 in mice results in renal cystic disease

  • Investigate skeletal and chondral development in appropriate models

• Quantitative approaches:

  • Measure ciliary length and frequency in affected tissues

  • Quantify colocalization of IFT140 with other IFT proteins

  • Assess changes in interaction strength using quantitative proteomics

What methodological approaches can be used to study the effects of IFT140 mutations?

To investigate how mutations in IFT140 affect protein function and contribute to disease:

How can IFT140 antibodies be used in multi-omics research approaches?

Integrate IFT140 antibodies into multi-omics research strategies:

• Proteomics integration:

  • Combine immunoprecipitation with mass spectrometry (IP-MS) to identify the complete IFT140 interactome

  • Use SILAC or TMT labeling for quantitative comparison between conditions

  • Perform cross-linking mass spectrometry (XL-MS) to map interaction interfaces

• Transcriptomics correlation:

  • Correlate IFT140 protein levels (detected by antibodies) with transcript expression

  • Investigate regulatory mechanisms controlling IFT140 expression

  • Study transcriptional changes in response to IFT140 mutations or depletion

• Spatial biology applications:

  • Use IFT140 antibodies in multiplexed immunofluorescence or imaging mass cytometry

  • Apply spatial transcriptomics alongside protein localization

  • Investigate tissue-specific differences in IFT140 function and interactome

• Systems biology perspective:

  • Map IFT140 into cellular signaling networks

  • Model how disruptions in IFT140 propagate through interacting systems

  • Identify potential therapeutic targets within the network

• Single-cell approaches:

  • Combine single-cell proteomics with IFT140 antibody detection

  • Investigate cell-to-cell variability in cilia formation and function

What are the important considerations when designing experiments to study context-specific functions of IFT140?

Design experiments that capture the context-specific functions of IFT140:

• Tissue-specific considerations:

  • Different tissues show varying IFT140 expression and potentially different interactomes

  • Consider using tissue-specific models (organoids, conditional knockouts)

  • Validated tissues for IFT140 antibody testing include testis, skeletal muscle, and cell lines like HeLa, HepG2, and C2C12

• Developmental timing:

  • IFT140 may have stage-specific functions during development

  • Design time-course experiments covering relevant developmental stages

  • Consider inducible systems to manipulate IFT140 at specific timepoints

• Ciliary subtypes:

  • Primary cilia in different tissues may have specialized functions

  • Motile vs. non-motile cilia may utilize IFT140 differently

  • Design experiments to compare IFT140 function across ciliary subtypes

• Environmental conditions:

  • Cilia respond to environmental signals including flow, light, and chemical stimuli

  • Design experiments incorporating relevant physiological stimuli

  • Compare IFT140 localization and interactions under different conditions

• Disease context:

  • Consider how pathogenic conditions alter IFT140 function

  • Include disease-relevant mutations when studying protein interactions

  • Compare findings across different ciliopathy models to identify common mechanisms