CLPT1 Antibody

What is CLPTM1 and what are its primary biological functions?

CLPTM1 (Cleft Lip and Palate Associated Transmembrane Protein 1) is a transmembrane protein with a calculated molecular weight of approximately 62 kDa, though it typically appears between 65-75 kDa in Western blots due to post-translational modifications . It is also sometimes referred to as "putative lipid scramblase CLPTM1" in the literature . The protein is encoded by the CLPTM1 gene (NCBI Gene ID: 1209) .

While initially identified in association with cleft lip and palate development, CLPTM1 has broader biological functions that researchers continue to investigate. In plants, CLPTM1 homologs (ClpT1 and ClpT2) are associated with the ClpPR protease and show functional redundancy, as demonstrated by compensatory responses when either gene is knocked out . These proteins appear to be crucial for normal biological function, as evidenced by their conservation and the compensatory mechanisms observed when one is depleted.

For optimal CLPTM1 detection, sample preparation should be tailored to the specific application. In Western blotting, COS7 cell lysates have demonstrated clear detection of CLPTM1 . For brain tissue samples from mouse and rat models, careful homogenization in appropriate lysis buffers with protease inhibitors is essential for maintaining protein integrity .

For immunohistochemistry applications on brain tissue, antigen retrieval is critical. The recommended protocol involves using TE buffer at pH 9.0, though citrate buffer at pH 6.0 may serve as an alternative . Proper fixation and embedding procedures should be followed to preserve tissue architecture while maintaining epitope accessibility.

How should CLPTM1 antibodies be stored to maintain reactivity?

CLPTM1 antibodies require specific storage conditions to maintain optimal reactivity:

• Temperature: Store at -20°C for long-term stability .

• Buffer composition: Typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3-7.4 .

• Handling: Upon delivery, aliquot the antibody to minimize freeze-thaw cycles, which can degrade antibody performance .

• Stability: When properly stored, the antibodies remain stable for approximately one year after shipment .

• Thawing: Thaw aliquots at room temperature and briefly centrifuge before use to collect all material.

For smaller volume products (20μl), some manufacturers include 0.1% BSA as a stabilizing agent, which does not interfere with standard applications .

How can researchers optimize Western blot protocols specifically for CLPTM1 detection?

Optimizing Western blot protocols for CLPTM1 requires attention to several critical parameters:

• Sample preparation: Complete cell lysis is essential. Use RIPA buffer supplemented with protease inhibitors. For brain tissue samples, which show strong CLPTM1 expression, homogenization should be thorough but gentle to preserve protein integrity .

• Protein loading: Load 20-40μg of total protein per lane for cell lysates. For tissues with lower CLPTM1 expression, increasing to 50-60μg may improve detection.

• Gel percentage: Use 8-10% SDS-PAGE gels for optimal resolution around the 65-75 kDa range where CLPTM1 is detected .

• Transfer conditions: Semi-dry transfer at 15V for 15 minutes followed by 20V for 45 minutes with PVDF membrane (0.45μm pore size) yields consistent results.

• Blocking conditions: 5% non-fat milk in TBST for 1 hour at room temperature minimizes background while preserving epitope accessibility.

• Antibody incubation: Dilute primary antibody between 1:500-1:2000 in TBST with 5% BSA and incubate overnight at 4°C . After washing, use compatible HRP-conjugated secondary antibodies (anti-rabbit IgG) at 1:5000 dilution for 1 hour at room temperature .

• Detection method: Enhanced chemiluminescence (ECL) systems provide sufficient sensitivity for CLPTM1 detection in most samples. Exposure times of 1-5 minutes are typically adequate.

• Controls: Include positive controls such as mouse or rat brain tissue lysates, which consistently show CLPTM1 expression . Negative controls should include samples where the primary antibody is omitted or pre-absorbed with the immunizing peptide .

What strategies can address non-specific binding issues when using CLPTM1 antibodies?

Non-specific binding can complicate interpretation of CLPTM1 immunodetection. Researchers can implement several strategies to minimize this issue:

• Antibody validation: Verify antibody specificity using knockout or knockdown models. In CLPTM1 studies, the clpt1-2 knockout shows complete absence of the normal CLPTM1 band, confirming antibody specificity .

• Epitope competition: Pre-absorb the antibody with the immunizing peptide (for example, the synthetic peptide derived from human CLPTM1 amino acids 200-249) to confirm binding specificity.

• Optimized blocking: Extend blocking time to 2 hours at room temperature using 5% BSA in TBST rather than milk for probing phosphorylated epitopes.

• Modified washing: Increase washing stringency by adding 0.1% SDS to TBST washing buffer and extending washing times to 15 minutes per wash, with at least 4 washes.

• Antibody dilution optimization: Further dilute antibodies beyond the recommended range if background remains high. For CLPTM1, testing dilutions up to 1:5000 for Western blot may be beneficial in high-background scenarios.

• Cross-reactivity assessment: Be aware that CLPTM1 antibodies may detect both normal and modified forms of the protein. The anti-CLPT1 antibody detected a higher molecular mass band (~2 kDa higher) in specific knockout backgrounds due to read-through translation , which may complicate interpretation if not properly accounted for.

How can researchers accurately quantify CLPTM1 expression levels?

Accurate quantification of CLPTM1 expression requires rigorous experimental design and analysis:

• Normalization strategy: Always normalize CLPTM1 signal to multiple housekeeping proteins (GAPDH, β-actin, and α-tubulin) to account for loading variations and ensure reliable quantification.

• Standard curve generation: For absolute quantification, prepare a standard curve using recombinant CLPTM1 protein at known concentrations.

• Image acquisition: Use a digital imaging system with a linear dynamic range for densitometry. Avoid film exposure, which can saturate signals and compromise quantitative analysis.

• Replication requirements: Perform at least three biological replicates and three technical replicates to account for biological variability and technical noise.

• Expression changes: For studying expression changes, be aware that compensatory responses may occur between related proteins. For example, ClpT1 protein levels increased ~3-fold in clpt2-1 mutants, while ClpT2 increased ~4-fold in clpt1-2 mutants .

• Statistical analysis: Apply appropriate statistical tests (ANOVA with post-hoc tests for multiple comparisons, t-tests for two-group comparisons) and report exact p-values along with effect sizes.

• Verification through multiple methods: Confirm Western blot quantification results with orthogonal techniques such as qRT-PCR for mRNA levels or mass spectrometry for protein quantification.

What factors influence the apparent molecular weight variations of CLPTM1 in experimental data?

CLPTM1 commonly appears at variable molecular weights across different experimental conditions and tissues:

• Expected molecular weight range: The calculated molecular weight of CLPTM1 is 62 kDa, but it typically appears between 65-75 kDa on Western blots . This discrepancy can be attributed to several factors:

• Post-translational modifications: Glycosylation, phosphorylation, and other modifications can significantly alter migration patterns. The transmembrane nature of CLPTM1 suggests potential glycosylation.

• Alternative splicing: Different CLPTM1 isoforms may exist. Studies have documented a higher molecular mass band (~2 kDa higher) in specific genetic backgrounds due to read-through translation when the normal stop codon is disrupted .

• Sample preparation effects: Incomplete denaturation or reduction can cause anomalous migration. Always ensure complete sample denaturation (95°C for 5 minutes in Laemmli buffer with β-mercaptoethanol).

• Gel percentage considerations: The resolution of proteins around CLPTM1's molecular weight is optimal in 8-10% acrylamide gels. Higher percentage gels may cause compression of bands and inaccurate molecular weight estimation.

• Marker calibration: Always use pre-stained protein standards that encompass the 50-100 kDa range for accurate molecular weight determination of CLPTM1.

What controls are essential when performing immunoprecipitation with CLPTM1 antibodies?

Immunoprecipitation of CLPTM1 requires comprehensive controls to ensure valid and interpretable results:

• Input control: Always reserve 5-10% of the pre-IP lysate to confirm target protein presence in the starting material.

• Negative antibody control: Perform parallel IP with non-specific IgG from the same host species (rabbit IgG isotype controls such as A82272 or A17360) to identify non-specific binding.

• Beads-only control: Include a sample with beads but no antibody to detect proteins that bind directly to the matrix.

• Reverse IP validation: When possible, confirm interactions by performing IP with antibodies against putative interaction partners and blotting for CLPTM1.

• Knockout/knockdown validation: The ultimate specificity control is performing IP in samples where CLPTM1 has been depleted through genetic approaches.

• Peptide competition: Pre-incubate the IP antibody with the immunizing peptide to block specific binding sites and confirm specificity of pulled-down bands.

• Cross-linking considerations: For transient or weak interactions, consider using chemical cross-linkers before cell lysis, but be aware this may affect epitope recognition.

For CLPTM1 immunoprecipitation, validated protocols have been established for A549 cells using 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate .

How can researchers validate compensatory mechanisms between CLPTM1 and related proteins?

Studies have revealed important compensatory relationships between CLPTM1 and related proteins, particularly in plant models:

• Transcriptional analysis: RT-PCR or RNA-Seq can detect transcriptional compensation. In clpt1 mutants, CLPT2 transcript levels increased ~1.6-fold, while in clpt2 mutants, CLPT1 transcripts showed similar modest increases .

• Protein-level assessment: Western blot analysis reveals more dramatic compensation at the protein level than at the transcript level. ClpT1 protein levels increased ~3-fold in clpt2-1 mutants, while ClpT2 increased ~4-fold in clpt1-2 mutants . This suggests post-transcriptional regulatory mechanisms are involved in compensation.

• Double knockout studies: Generate double mutants lacking both proteins to assess functional redundancy. Complete loss of both proteins would eliminate compensatory mechanisms and potentially reveal more severe phenotypes.

• Rescue experiments: Introduce wild-type genes or tagged variants into single or double mutant backgrounds to determine if function can be restored and to what extent.

• Domain-swapping experiments: Create chimeric proteins combining domains from related proteins to identify which regions are responsible for functional redundancy and compensation.

• Temporal analysis: Track expression changes over developmental time to determine if compensation is immediate or develops gradually, providing insights into regulatory mechanisms.

What are the critical factors for successful immunohistochemistry using CLPTM1 antibodies?

Immunohistochemistry with CLPTM1 antibodies requires careful attention to tissue processing and staining protocols:

• Tissue fixation: Optimal fixation uses 4% paraformaldehyde for 24-48 hours, followed by proper paraffin embedding. Over-fixation can mask epitopes and reduce antibody binding.

• Antigen retrieval method: For CLPTM1 detection in brain tissue, TE buffer at pH 9.0 is recommended, though citrate buffer at pH 6.0 may be used as an alternative . Heat-induced epitope retrieval (pressure cooker or microwave heating) typically yields better results than enzymatic methods.

• Antibody dilution: Start with a 1:100 dilution for initial optimization, then adjust within the recommended 1:50-1:500 range based on signal-to-noise ratio .

• Incubation conditions: Overnight incubation at 4°C generally produces better results than shorter incubations at room temperature.

• Detection system selection: For low-abundance targets, use high-sensitivity detection systems such as polymer-based HRP systems or tyramide signal amplification.

• Counterstaining considerations: Use light hematoxylin counterstaining to avoid obscuring specific CLPTM1 signal, particularly in tissues with expected cytoplasmic or membrane staining patterns.

• Negative controls: Include sections where primary antibody is omitted or replaced with non-specific IgG. Ideally, include tissue from knockout models when available.

• Positive controls: Mouse brain tissue has been validated as a reliable positive control for CLPTM1 immunohistochemistry .

How do researchers interpret unexpected band patterns in CLPTM1 Western blots?

Unexpected band patterns in CLPTM1 Western blots require careful interpretation and follow-up experiments:

• Higher molecular weight bands: Bands at ~78-80 kDa (approximately 2 kDa higher than expected) may represent alternative translation products. In clpt1-2 mutants, read-through translation due to disruption of the normal stop codon results in a higher mass band, confirmed by sequencing and RT-PCR .

• Lower molecular weight bands: These often represent degradation products or proteolytic fragments. Minimize by using fresh samples, maintaining samples at 4°C during preparation, and adding protease inhibitor cocktails to lysis buffers.

• Multiple bands: May indicate different isoforms, post-translational modifications, or non-specific binding. Validate using peptide competition assays, where pre-incubation of the antibody with immunizing peptide should eliminate specific bands.

• Tissue-specific patterns: CLPTM1 expression patterns may vary between tissues. Brain tissue consistently shows strong expression and can serve as a positive control .

• Verification approaches:

  • Immunoprecipitation followed by mass spectrometry to confirm protein identity

  • RNA interference to confirm band specificity (bands should decrease with target knockdown)

  • Use of multiple antibodies targeting different epitopes of CLPTM1 to confirm band identity

What methodological approaches can resolve contradictory results between different CLPTM1 antibodies?

When different CLPTM1 antibodies produce contradictory results, systematic troubleshooting is required:

• Epitope mapping comparison: Compare the immunogens used to generate each antibody. For example, one antibody targets the central region of CLPTM1 , while another targets amino acids 200-249 . Different epitopes may be differentially accessible depending on protein conformation or interactions.

• Cross-validation strategy: Apply multiple detection methods:

  • Use at least three different antibodies targeting different epitopes

  • Compare results from transcriptional analysis (RT-PCR, RNA-Seq)

  • Employ mass spectrometry for unbiased protein identification

• Domain-specific antibody testing: If contradictory results persist, develop domain-specific antibodies to target different regions of CLPTM1 and compare their detection patterns.

• Genetic validation: Generate CRISPR knockout or knockdown models to confirm antibody specificity. All specific bands should be absent or reduced in knockout/knockdown samples.

• Antibody validation reporting: Document comprehensive validation data including:

  • Positive and negative controls used

  • Full blot images showing all bands

  • Peptide competition results

  • Knockout/knockdown validation

  • Cross-reactivity assessment with similar proteins

• Methodological standardization: Standardize protocols across laboratories to eliminate technical variables that might contribute to contradictory results.

How can CLPTM1 antibodies be optimized for multiplex immunofluorescence applications?

Multiplexing CLPTM1 detection with other proteins requires careful optimization:

• Antibody host selection: Choose primary antibodies raised in different host species to allow simultaneous detection. For CLPTM1, rabbit polyclonal antibodies are common , so pair with mouse, rat, or goat antibodies against other targets.

• Fluorophore selection: Select fluorophores with minimal spectral overlap. For rabbit anti-CLPTM1 antibodies, consider secondary antibodies conjugated to:

  • Alexa Fluor 488 (green)

  • Cy3 (red)

  • Alexa Fluor 647 (far-red)

• Sequential staining protocol: If antibody host constraints exist, use sequential staining with complete stripping or fluorophore inactivation between rounds.

• Cross-reactivity testing: Perform single-staining controls to ensure secondary antibodies don't cross-react with primary antibodies from different species.

• Signal separation validation: Perform co-localization analysis on control samples with known expression patterns to validate signal separation.

• Optimal fixation methods: For multiplex immunofluorescence, 2% PFA for 10-15 minutes typically preserves both antigenicity and cellular architecture better than longer fixation times.

• Signal amplification considerations: For low-abundance targets, consider tyramide signal amplification (TSA) which can increase detection sensitivity 10-100 fold.

What approaches can elucidate CLPTM1 protein interactions and functional complexes?

Understanding CLPTM1's protein interactions requires specialized approaches:

• Co-immunoprecipitation optimization: Use mild lysis conditions (1% NP-40 or 0.5% CHAPS instead of stronger detergents) to preserve protein-protein interactions. Cross-linking with low concentrations (0.5-1%) of formaldehyde before lysis can stabilize transient interactions.

• Proximity labeling methods:

  • BioID: Fuse CLPTM1 to a biotin ligase (BirA*) to biotinylate proteins in close proximity

  • APEX2: Fuse CLPTM1 to APEX2 peroxidase for proximity-based labeling

  • These approaches can identify interaction partners without requiring stable associations

• Mass spectrometry analysis: After immunoprecipitation, use tandem mass spectrometry to identify co-precipitating proteins. Compare results to IgG controls to filter out non-specific interactions.

• Yeast two-hybrid screening: While challenging for transmembrane proteins like CLPTM1, modified split-ubiquitin Y2H systems can be effective for identifying interactions.

• FRET/BRET analysis: For suspected interactions, create fluorescent or bioluminescent fusion proteins and measure energy transfer to confirm close proximity in living cells.

• Functional complex analysis: In plant systems, ClpT1 and ClpT2 associate with the ClpPR protease . Similar approaches using gradient centrifugation or native PAGE can identify CLPTM1-containing complexes in mammalian systems.

• Genetic interaction screens: CRISPR-based screens can identify genes that show synthetic lethality or enhanced phenotypes when combined with CLPTM1 depletion, suggesting functional relationships.

What are the best practices for studying post-translational modifications of CLPTM1?

Post-translational modifications (PTMs) of CLPTM1 can be studied using these approaches:

• Phosphorylation detection:

  • Use phospho-specific antibodies if available

  • Treat samples with lambda phosphatase to confirm phosphorylation status

  • Perform immunoprecipitation followed by Western blotting with anti-phospho-Ser/Thr/Tyr antibodies

• Glycosylation analysis:

  • Treat lysates with PNGase F or Endo H to remove N-linked glycans before Western blotting

  • Observe mobility shifts that indicate glycosylation

  • For transmembrane proteins like CLPTM1, N-glycosylation is common and may affect apparent molecular weight

• Ubiquitination studies:

  • Immunoprecipitate CLPTM1 and blot for ubiquitin

  • Alternatively, immunoprecipitate ubiquitinated proteins using anti-ubiquitin antibodies and blot for CLPTM1

  • Treat cells with proteasome inhibitors (MG132) to accumulate ubiquitinated species

• Mass spectrometry approaches:

  • Enrich for CLPTM1 by immunoprecipitation

  • Perform tryptic digestion and analyze by LC-MS/MS

  • Use neutral loss scanning for phosphorylation

  • Use lectins or HILIC chromatography to enrich glycopeptides

• Site-directed mutagenesis:

  • Mutate predicted modification sites (Ser/Thr/Tyr for phosphorylation, Asn for N-glycosylation)

  • Express wild-type and mutant forms to compare molecular weights and functional properties

• Temporal dynamics:

  • Study modification patterns after relevant stimuli or during different cell cycle phases

  • Use pulse-chase approaches to track the lifetime of modifications