CLTCL1 Antibody

What is CLTCL1 and what are the key characteristics of antibodies targeting it?

CLTCL1 (also known as CHC22, CLH22, CLTCL, or CLTD) encodes clathrin heavy chain-like 1, a protein with a calculated molecular weight of 187 kDa, though it typically appears around 180 kDa on western blots . The gene has ID 8218 and produces a protein involved in intracellular membrane trafficking . CLTCL1 antibodies are available as rabbit polyclonal (such as Prestige Antibodies powered by Atlas Antibodies) and mouse monoclonal formats, with demonstrated reactivity against human samples . These antibodies have been extensively validated through multiple techniques including western blotting, immunohistochemistry, and immunofluorescence, with data accessible through resources like the Human Protein Atlas .

What are the validated applications for CLTCL1 antibodies in research settings?

CLTCL1 antibodies have been successfully validated for multiple experimental applications:

Additionally, some CLTCL1 antibodies have been tested against tissue arrays comprising 44 normal human tissues and 20 common cancer types, demonstrating their utility in examining protein expression patterns across diverse tissue contexts .

How should researchers optimize CLTCL1 antibody protocols for neural tissue studies?

When investigating CLTCL1 in neural tissues, researchers should consider several factors based on the protein's developmental expression pattern. CLTCL1 shows significant upregulation in the developing human brain, suggesting an important neurodevelopmental role . For immunohistochemistry of neural tissues, optimization should include:

• Antigen retrieval optimization: Test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) methods

• Antibody dilution: Start with 1:50-1:500 range and titrate for optimal signal-to-noise ratio

• Developmental timing considerations: Expression levels vary during developmental stages, with CHC22 showing downregulation upon neural differentiation in human iPSC-derived nociceptors and TRKB-dependent SH-SY5Y cells

• Counter-staining with neural markers: Include neural precursor and differentiation markers to contextualize CHC22 expression patterns

Note that knockdown studies have shown that CHC22 reduction can induce neurite outgrowth in neural precursor cells, suggesting a regulatory role in neural differentiation .

What controls are essential when validating CLTCL1 antibody specificity?

Proper validation of CLTCL1 antibodies requires several critical controls:

• Positive tissue controls: Human skeletal muscle tissue has demonstrated reliable positive staining and should be included as a positive control

• Cross-reactivity assessment: CLTCL1 antibodies should be validated against CLTC (the related clathrin heavy chain), as some antibodies are specifically designed not to cross-react with CLTC

• Recombinant protein arrays: High-quality antibodies have been tested against arrays of 364 human recombinant protein fragments to confirm specificity

• Genetic models: Knockdown/knockout cell lines provide definitive controls; transient siRNA knockdown of CLTCL1 followed by antibody staining can confirm specificity

• Variant-specific validation: When studying the M1316 versus V1316 variants, controls should include cells expressing each variant to ensure antibody detection is not affected by this polymorphism

How does the CHC22 protein function in GLUT4 trafficking and what methodological approaches are recommended for studying this pathway?

CHC22 clathrin (encoded by CLTCL1) plays a key role in intracellular membrane trafficking of the insulin-responsive GLUT4 glucose transporter, which is critical for post-prandial clearance of glucose from human blood . To study this pathway:

• Cellular models: Human adipocytes or muscle cells expressing endogenous GLUT4 are preferred systems

• Trafficking assays: Researchers should employ:

  • Surface biotinylation assays to measure GLUT4 translocation

  • Co-immunoprecipitation to identify CHC22 interaction partners

  • Confocal microscopy with CLTCL1 antibodies to track vesicle movement

• Variant considerations: Two high-frequency variants of CHC22 (M1316 and V1316) show functional differences in GLUT4 trafficking capacity. The V1316 variant appears only in humans, arising 500-50 KYA, with higher frequency in farming versus hunter-gatherer populations, suggesting metabolic adaptation

• Knockout/rescue experiments: CLTCL1 knockdown followed by rescue with wild-type or mutant (e.g., E330K) constructs can demonstrate functional differences and specificity

• Evolutionary context: Studies should consider the variable presence of CLTCL1 across vertebrates (independently lost in nine lineages), while the parent CLTC gene is universally retained

What is currently understood about CLTCL1's role in disease pathology and what experimental approaches are recommended for these investigations?

CLTCL1 has been implicated in several pathological conditions with specific methodological considerations for each:

• Neurodevelopmental disorders: A homozygous CLTCL1 mutation (c.988G>A, p.E330K) has been associated with an autosomal recessive disorder affecting neural development . Research approaches should include:

  • Neurite outgrowth assays in neural precursor cells

  • Stable expression of wild-type vs. mutant CHC22 in rescue experiments

  • Assessment of downstream signaling pathways affected by CHC22 dysfunction

• Cancer progression: CLTCL1 down-regulation appears to be an early event in ductal carcinoma progression, preceding invasiveness . Investigations should employ:

  • Laser microdissection to isolate pure populations of epithelial cells from different disease stages

  • RT-qPCR to quantify expression changes

  • Correlation of expression with clinicopathological features and invasion markers

• Metabolic disorders: Given CHC22's role in GLUT4 trafficking, studies into metabolic conditions should include:

  • Genotyping for M1316V polymorphism in human cohorts

  • Glucose tolerance tests correlated with genotype

  • Functional trafficking assays comparing variant performance

How can researchers effectively distinguish between CHC22 (CLTCL1) and the related CHC17 (CLTC) in experimental systems?

Distinguishing between the two clathrin heavy chains requires careful experimental design:

• Antibody selection: Use antibodies specifically validated for lack of cross-reactivity between CLTCL1 and CLTC. For example, antibody 66274-1-Ig has been specifically tested and shows no cross-reaction to CLTC

• Expression pattern analysis: CHC22 and CHC17 show different tissue distributions. While CHC17 is ubiquitously expressed and mediates endocytosis and other housekeeping membrane traffic pathways, CHC22 has a more restricted expression pattern

• Functional assays:

  • CHC22 specifically functions in GLUT4 trafficking

  • CHC17 functions in general endocytosis

• Evolutionary context: All vertebrates retain CLTC (encoding CHC17), while CLTCL1 (encoding CHC22) has been independently lost in nine vertebrate lineages, providing a comparative experimental framework

• Knockout strategies: Selective knockdown of each gene can help disambiguate their functions, but compensatory mechanisms should be considered

What are the optimal sample preparation methods for detecting CLTCL1/CHC22 in different experimental contexts?

Sample preparation should be tailored to the experimental context:

• Western blotting:

  • Expected molecular weight: ~180 kDa (observed) vs 187 kDa (calculated)

  • Sample buffer: PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 is recommended for antibody storage

  • Positive controls: HeLa, HEK-293, and Jurkat cell lysates have shown reliable detection

• Immunohistochemistry:

  • Fixation: Typically formalin-fixed paraffin-embedded tissues

  • Antigen retrieval: TE buffer at pH 9.0 is preferred, though citrate buffer at pH 6.0 is an alternative

  • Skeletal muscle serves as a reliable positive control tissue

• Immunofluorescence:

  • Cell types: HeLa cells provide a good positive control

  • Fixation: 4% paraformaldehyde followed by permeabilization is standard

  • When studying neural differentiation, consider examining CHC22 expression at different timepoints, as downregulation occurs upon neural differentiation

• Long-term storage: Store antibodies at -20°C for stability. For the 20μl size, products contain 0.1% BSA. Aliquoting is unnecessary for -20°C storage

How should researchers approach studies involving CLTCL1 mutations and their functional effects?

When investigating CLTCL1 mutations such as the pathogenic E330K variant:

• Mutation validation strategies:

  • PCR amplification and sequencing of the region containing the mutation (e.g., exon 7 for c.988G>A)

  • Restriction fragment length polymorphism (RFLP) analysis if the mutation creates or destroys a restriction site

  • Segregation analysis in affected families to confirm inheritance patterns

• Functional assessment:

  • Create stably expressing cell lines with wild-type or mutant constructs

  • Transiently transfect cells followed by functional assays after 48-72 hours

  • For selection of stable transfectants, G418 at 600 μg/ml has been successful

• Expression challenges: Wild-type CHC22 may be more difficult to stably express than mutant versions, requiring screening of multiple clones to find comparable expression levels

• Rescue experiments: Knockdown endogenous CLTCL1 using siRNA, then rescue with siRNA-resistant wild-type or mutant constructs to assess functional differences

• Cellular readouts: For the E330K mutation, neurite outgrowth serves as a functional readout, with wild-type CHC22 blocking outgrowth in retinoic acid-treated cells, while the mutant fails to do so

What approaches are recommended for studying CHC22 variant differences in population and evolutionary contexts?

To investigate the evolutionary and population differences in CHC22 variants:

• Population genetic analyses:

  • Genotyping for the M1316V polymorphism across diverse human populations

  • Statistical analysis shows extensive allelic diversity for CLTCL1 compared to CLTC in humans and chimpanzees

  • Compare frequencies between different human populations, noting higher V1316 frequency in farming versus hunter-gatherer populations

• Evolutionary analysis methodologies:

  • Phylogenetic approaches to track gene loss/retention across vertebrate lineages

  • Statistical tests for purifying selection (dN/dS ratios) over phylogenetic timescales

  • Sequence alignment of homologous regions across species to identify conserved domains

• Functional differentiation:

  • Directly compare M1316 and V1316 variants in GLUT4 trafficking assays

  • Use structural modeling to predict functional differences between variants

  • Correlate with physiological measurements in human subjects of different genotypes

• Ancient DNA analysis: The V1316 variant has been dated to 500-50 KYA through analysis of archaic and ancient human DNA