IFT172 Antibody, HRP conjugated

What is IFT172 and why is it significant in ciliary research?

Intraflagellar Transport 172 Homolog (IFT172) is a critical component of the intraflagellar transport (IFT) system, which is essential for assembly and maintenance of cilia and flagella, as well as ciliary motility and signaling functions . IFT172 functions as one of the peripheral components of the IFT-B complex and can be dissociated from the salt-resistant core subcomplex under high salt treatment . The protein is particularly significant in research because it represents an important element of a conserved molecular machinery that is fundamental to ciliary biology across diverse species, with percent identity confirmed across mammals, birds, and fish species . Mutations in IFT172 have been linked to ciliopathies, making antibodies against this protein valuable tools for investigating disease mechanisms.

What applications are most suitable for IFT172 antibodies?

IFT172 antibodies can be effectively employed in multiple experimental applications including:

• Western Blotting (WB): Effectively detects IFT172 protein at approximately 180-198 kDa

• Immunohistochemistry (IHC): Particularly effective in paraffin-embedded tissue sections with appropriate antigen retrieval methods

• Immunofluorescence (IF): Useful for cellular localization studies, particularly in ciliated cell types

• ELISA: Provides quantitative measurement of IFT172 protein levels

Each application requires specific optimization of antibody dilutions, with recommended ranges typically between 1:50-1:500 for IHC and IF applications, and 1:1000-1:4000 for Western blotting . HRP-conjugated variants are particularly advantageous for Western blotting and ELISA applications as they eliminate the need for secondary antibody incubation, reducing background and saving experimental time.

What species reactivity can I expect from IFT172 antibodies?

IFT172 antibodies exhibit broad cross-species reactivity due to the high conservation of this protein across diverse species. Based on BLAST analysis and empirical testing, antibodies raised against human IFT172 epitopes typically demonstrate reactivity with samples from:

This broad cross-reactivity makes IFT172 antibodies versatile tools for comparative biology research, though optimization may be required when working with more evolutionarily distant species .

How can I optimize detection specificity when using HRP-conjugated IFT172 antibodies in complex tissue samples?

When working with complex tissue samples, particularly those with high ciliary content such as testis, brain, or retinal tissues, several optimization strategies can enhance detection specificity:

• Antigen retrieval optimization: For IHC applications, test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) to determine optimal epitope exposure conditions. Test data indicates TE buffer at pH 9.0 provides superior results for testis tissue samples .

• Blocking optimization: When using HRP-conjugated antibodies directly, implement a multi-step blocking protocol:

  • Begin with standard protein blocking (BSA or serum)

  • Follow with peroxidase blocking (H₂O₂ treatment)

  • Consider additional avidin/biotin blocking if tissues contain endogenous biotin

• Dilution titration: Perform systematic titration within the recommended dilution range (starting at 1:50-1:500) to identify the optimal signal-to-noise ratio for your specific tissue .

• Validation controls: Include tissues from IFT172-knockout models or IFT172-depleted samples as negative controls to confirm specificity.

For tissues with high background potential, consider signal amplification systems compatible with HRP, such as tyramide signal amplification, while being mindful of potential increases in background signal.

What are the key considerations when validating protein interactions involving IFT172 using antibody-based approaches?

Validating protein-protein interactions involving IFT172 requires careful experimental design due to the complex architecture of the IFT-B complex. Based on contemporary research methodologies:

• Selecting appropriate interaction assays: Visible immunoprecipitation (VIP) assays have proven effective for studying IFT-B complex interactions . This approach allows visualization of complex protein interactions, including one-to-many and many-to-many interactions that are common in the IFT system.

• Considering salt conditions: IFT172 is one of the peripherally associated subunits of the IFT-B complex that dissociates under high salt conditions . Therefore, interaction studies should test multiple salt concentrations to differentiate between core and peripheral interactions.

• Addressing reciprocal validations: When using antibody-based approaches like co-immunoprecipitation, validate interactions in both directions. As noted in the literature, interactions should only be considered "positive" when signals are detected in reciprocal combinations of tagged proteins under identical conditions .

• Managing antibody cross-reactivity: When using HRP-conjugated antibodies in multiplex studies, be aware of potential cross-reactivity. Pre-adsorption against related proteins or using epitope-specific antibodies targeting distinct regions of IFT172 can minimize this issue.

• Expression level concerns: Protein expression levels can significantly impact interaction detection. As observed in VIP assays, "the expression level and/or stability of certain fluorescent fusion proteins are often affected by co-expressed proteins" . Similar considerations apply when using antibodies to detect native interactions.

How do HRP-conjugated IFT172 antibodies perform in detecting post-translational modifications compared to unconjugated variants?

Detection of post-translational modifications (PTMs) on IFT172 presents unique challenges that require consideration when selecting between HRP-conjugated and unconjugated antibodies:

• Epitope masking effects: HRP conjugation can potentially mask or alter antibody binding when the PTM site is proximal to the antibody epitope. When studying phosphorylation, SUMOylation, or ubiquitination of IFT172, unconjugated primary antibodies followed by secondary detection may provide better sensitivity and specificity.

• PTM-specific considerations:

  • For phosphorylation studies: Test both conjugated and unconjugated antibodies against samples treated with and without phosphatase inhibitors to confirm specificity.

  • For ubiquitination studies: HRP-conjugated antibodies may exhibit reduced sensitivity due to steric hindrance, particularly when detecting poly-ubiquitin chains.

• Signal amplification trade-offs: While HRP-conjugated antibodies offer direct detection, they provide less signal amplification compared to the two-step detection method using unconjugated primary and HRP-conjugated secondary antibodies. This becomes critical when detecting low-abundance PTM species.

• Multiplexing limitations: When studying multiple PTMs simultaneously, HRP-conjugated antibodies limit multiplexing options compared to unconjugated variants that can be paired with differently labeled secondary antibodies.

For challenging PTM detection scenarios, consider using unconjugated IFT172 antibodies combined with highly sensitive detection systems, reserving HRP-conjugated variants for standard protein detection applications.

What is the optimal protocol for using HRP-conjugated IFT172 antibodies in Western blotting?

The following optimized protocol has been developed based on experimental evidence with IFT172 antibodies:

Materials Required:

• HRP-conjugated IFT172 antibody

• PVDF or nitrocellulose membrane with transferred proteins

• Blocking buffer (5% non-fat dry milk or 3-5% BSA in TBST)

• TBS-T (TBS with 0.1% Tween-20)

• Enhanced chemiluminescence (ECL) substrate

Protocol:

• Sample Preparation: For optimal detection of IFT172, include testis tissue as a positive control when possible, as it shows consistent high expression of the target protein .

• Blocking: Block membrane in 5% non-fat dry milk or 3-5% BSA in TBST for 1 hour at room temperature.

• Primary Antibody Incubation: Dilute HRP-conjugated IFT172 antibody to 1:1000-1:4000 in blocking buffer. Incubate membrane overnight at 4°C with gentle rocking .

• Washing: Wash membrane 3-5 times with TBST, 5-10 minutes per wash.

• Detection: Apply ECL substrate according to manufacturer's instructions and detect signal.

Important Notes:

• Expected molecular weight for IFT172 is approximately 180-198 kDa .

• When troubleshooting, consider that the observed molecular weight (180 kDa) may differ slightly from the calculated weight (198 kDa) .

• For improved results with mouse or rat samples, brain and testis tissues yield optimal detection results .

How can I optimize immunohistochemistry protocols using HRP-conjugated IFT172 antibodies?

Optimized IHC Protocol for HRP-conjugated IFT172 Antibodies:

Materials:

• Paraffin-embedded tissue sections

• Xylene and graded alcohols

• Antigen retrieval buffer (TE buffer pH 9.0 recommended)

• 3% H₂O₂ in methanol

• Blocking buffer (10% normal serum in PBS)

• HRP-conjugated IFT172 antibody

• DAB substrate kit

• Hematoxylin counterstain

Protocol:

• Deparaffinization and Rehydration: Standard xylene and graded alcohol series.

• Antigen Retrieval: Heat-induced epitope retrieval using TE buffer pH 9.0 is strongly recommended based on experimental data with testis tissue samples. Alternatively, citrate buffer pH 6.0 may be used but may result in reduced signal intensity .

• Peroxidase Blocking: Incubate sections in 3% H₂O₂ in methanol for 10 minutes to block endogenous peroxidase activity.

• Protein Blocking: Block with 10% normal serum in PBS for 30 minutes at room temperature.

• Antibody Application: Dilute HRP-conjugated IFT172 antibody to 1:50-1:500 in blocking buffer. Incubate sections overnight at 4°C in a humidified chamber .

• Washing: Wash 3 times with PBS, 5 minutes per wash.

• Detection: Apply DAB substrate and monitor for color development. Counterstain with hematoxylin.

• Mounting: Dehydrate, clear, and mount with permanent mounting medium.

Optimization Guidance:

• Begin with 1:100 dilution and adjust based on signal intensity and background levels

• For ciliated tissues (testis, respiratory epithelium, brain), shorter antibody incubation times (2-4 hours) may be sufficient

• When staining tissues with high background potential, include an additional avidin/biotin blocking step

What are the best practices for troubleshooting weak or non-specific signals when using IFT172 antibodies?

When encountering signal issues with IFT172 antibodies, including HRP-conjugated variants, implement the following systematic troubleshooting approach:

For Weak Signal:

For Non-specific Signals:

Additional Validation Approaches:

• Compare staining patterns with unconjugated IFT172 antibodies targeting different epitopes

• Include competitive blocking controls using recombinant IFT172 antigen

• Confirm specificity using siRNA knockdown or CRISPR knockout samples

• For tissues with naturally high ciliary content, use adjacent non-ciliated tissues as internal negative controls

How can HRP-conjugated IFT172 antibodies be effectively utilized in ciliopathy research?

HRP-conjugated IFT172 antibodies provide valuable tools for investigating ciliopathies through several strategic applications:

• Diagnostic Biomarker Development:

  • HRP-conjugated antibodies enable high-throughput screening of tissue samples for altered IFT172 expression or localization

  • The direct conjugation allows for rapid protocol execution in clinical research settings

  • Particularly valuable for examining testis, brain, and retinal tissues where ciliary defects manifest prominently

• Therapeutic Response Monitoring:

  • Quantitative assessment of IFT172 localization and abundance following experimental treatments

  • Serial sampling to track treatment efficacy in pre-clinical models

  • Analysis of ciliary rescue in patient-derived cellular models

• Mechanistic Studies:

  • Investigation of IFT172's interactions within the IFT-B complex using techniques like proximity ligation assays

  • Examination of how mutations affect IFT172's integration into the peripheral subcomplex of IFT-B

  • Assessment of IFT172's proper localization to ciliary structures

• High-Content Screening Applications:

  • Development of automated image analysis workflows using HRP-based colorimetric detection

  • Screening of compound libraries for agents that correct IFT172 mislocalization

  • Multiplexed screening approaches combining HRP-conjugated IFT172 antibodies with markers of ciliary function

When designing ciliopathy research protocols, consider that IFT172 functions as part of a "weakly associated" peripheral component of the IFT-B complex that dissociates under high salt conditions, which may have implications for experimental design and sample preparation .

What are the emerging applications of IFT172 antibodies in developmental biology research?

Developmental biology research represents a frontier application area for IFT172 antibodies, with several key applications:

• Developmental Timing of Ciliogenesis:

  • HRP-conjugated IFT172 antibodies enable precise temporal tracking of when functional IFT machinery assembles during embryonic development

  • The broad cross-species reactivity of these antibodies (human, mouse, rat, and multiple model organisms) facilitates comparative developmental studies

  • Direct comparison of IFT172 expression patterns across species with different percent identity: mammals (100%), fish (92%), and birds (85%)

• Tissue-Specific Ciliary Specialization:

  • Investigation of tissue-specific modifications of IFT172 function during organogenesis

  • Comparison of IFT172 localization and abundance between primary cilia of different developing tissues

  • Correlation of IFT172 patterns with cell differentiation markers

• Developmental Phenotyping:

  • Characterization of ciliary defects in developmental models using IFT172 as a diagnostic marker

  • Assessment of IFT-B complex integrity during organ development

  • Analysis of how developmental signaling pathways influence IFT172 function and localization

• Stem Cell Differentiation:

  • Monitoring ciliary formation during directed differentiation protocols

  • Verification of proper IFT machinery assembly in engineered tissues

  • Quality control assessment of ciliated cell types derived from stem cells

When applying these approaches to developmental studies, researchers should note that IFT172 functions within a complex protein interaction network that includes other IFT-B components. The protein interactions map derived from VIP assays provides guidance on how IFT172 integrates with other components of the intraflagellar transport machinery .

How do storage and handling conditions affect the performance of HRP-conjugated IFT172 antibodies?

Proper storage and handling are critical for maintaining the functional integrity of HRP-conjugated IFT172 antibodies:

Optimal Storage Conditions:

• Store at -20°C in small aliquots to minimize freeze-thaw cycles

• Stability maintained for approximately one year when stored properly

• For dilute working solutions, store at 4°C for no more than one week

Storage Buffer Considerations:

• Typical storage buffer contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Note: Sodium azide can inhibit HRP activity, so ensure thorough washing before detection steps

• For working dilutions, prepare fresh in appropriate application buffer without azide

Stability Factors:

• HRP conjugation typically provides greater stability compared to fluorescent dye conjugates

• Avoid repeated freeze-thaw cycles which can lead to aggregate formation and reduced activity

• Protect from prolonged exposure to light, particularly during storage

Performance Monitoring:

• Include positive control samples (testis or brain tissue) in experiments to confirm antibody performance

• Monitor for changes in background signal or detection sensitivity as indicators of potential deterioration

• Consider implementing regular quality control testing for antibodies stored longer than 6 months

For maximum performance consistency, particularly in longitudinal studies, implementing standardized handling protocols across all laboratory members is strongly recommended.

What are the comparative advantages and limitations of HRP-conjugated versus unconjugated IFT172 antibodies?

When selecting between HRP-conjugated and unconjugated IFT172 antibodies, researchers should consider these comparative advantages and limitations:

Application-Specific Recommendations:

• For routine Western blotting of abundant proteins: HRP-conjugated offers workflow efficiency

• For detection of low-abundance targets: Unconjugated with amplification systems provides better sensitivity

• For co-localization studies: Unconjugated provides better multiplexing options

• For high-throughput screening: HRP-conjugated reduces protocol complexity and time

The choice between formats should be guided by the specific experimental requirements, target abundance, and desired downstream applications.

What are the current research frontiers involving IFT172 that would benefit from advanced antibody applications?

Several emerging research areas involving IFT172 would benefit significantly from advanced antibody applications:

• Single-Cell Ciliary Proteomics:

  • Application of highly specific IFT172 antibodies for immunoprecipitation followed by mass spectrometry

  • Investigation of cell-type specific IFT172 interaction partners in specialized ciliary structures

  • Analysis of how the IFT-B complex composition varies across different tissues and developmental stages

• Ciliopathy Patient Stratification:

  • Development of diagnostic protocols using IFT172 antibodies to identify specific mechanistic defects

  • Correlation of IFT172 abnormalities with clinical phenotypes

  • Classification of ciliopathies based on IFT-B complex integrity rather than end-organ effects

• Therapeutic Target Validation:

  • Use of HRP-conjugated IFT172 antibodies in high-throughput screens for compounds that correct trafficking defects

  • Monitoring IFT172 dynamics in response to candidate therapeutic interventions

  • Development of patient-derived organoid models with trackable IFT172 function

• Environmental Impact on Ciliary Function:

  • Investigation of how environmental factors affect IFT172 localization and function

  • Analysis of post-translational modifications on IFT172 in response to cellular stressors

  • Examination of ciliary remodeling mechanisms under changing environmental conditions

These research frontiers will benefit from continued refinement of antibody technologies, including development of conformation-specific antibodies that can distinguish between IFT172's free form versus its integrated state within the IFT-B complex .

How can researchers optimize experimental design when studying IFT172 in the context of the broader IFT-B complex?

When investigating IFT172 within the context of the broader IFT-B complex, researchers should implement these optimization strategies: