CLN5 Antibody, HRP conjugated

What is CLN5 and why is it important in neurological research?

CLN5 (Ceroid-Lipofuscinosis Neuronal protein 5) functions as a bis(monoacylglycero)phosphate synthase that catalyzes the synthesis of bis(monoacylglycero)phosphate (BMP) via transacylation of lysophosphatidylglycerol molecules . Mutations in the CLN5 gene cause variant late-infantile NCL, with disease onset typically between 4-7 years of age . CLN5 is ubiquitously expressed in most tissues, with expression in both neuronal and glial cells within the brain . Its importance in neurological research stems from its critical role in lysosomal function, where mutations lead to accumulation of autofluorescent storage material in lysosomes in the brain and peripheral tissues . Understanding CLN5 function provides insights into lysosomal biology, protein trafficking, and neurodegenerative disease mechanisms.

What applications are CLN5 antibodies suitable for?

According to the available data, CLN5 antibodies have been validated for multiple research applications:

CLN5 antibodies have been successfully used in human, mouse, and rat samples . For optimal results in each application, researchers should consider the specific epitope recognition and validation data provided by the manufacturer.

What is the molecular weight profile of CLN5 protein and how does this affect antibody detection?

CLN5 exhibits variable molecular weights due to extensive post-translational modifications, particularly glycosylation:

• Cell-free translation studies have identified CLN5 forms at 47, 44, 41, and 39 kDa

• In cellular expression systems, CLN5 appears primarily as a ~60 kDa glycoprotein

• When deglycosylated, CLN5 appears as a ~38 kDa protein

• The predicted molecular weight of the mature 358 amino acid CLN5 is approximately 38 kDa

Researchers should expect to observe different molecular weight bands depending on cell type, expression system, and glycosylation status. In HEK293FT cells and human iPSCs, for example, CLN5 appears predominantly as a ~60 kDa glycosylated form that reduces to ~38 kDa after deglycosylation . This variability should be considered when interpreting Western blot results with CLN5 antibodies.

How do I optimize Western blot protocols for detecting differentially glycosylated forms of CLN5?

Based on the research data, optimizing Western blots for CLN5 requires careful consideration of sample preparation and analysis:

• Sample preparation:

  • For total CLN5 detection, use standard lysis buffers with protease inhibitor cocktails

  • To analyze glycosylation patterns, prepare parallel samples for enzymatic deglycosylation

• Deglycosylation analysis:

  • Treat protein extracts with EndoH (sensitive to high-mannose oligosaccharides) and PNGaseF (removes all N-linked glycans)

  • Include untreated controls for comparison

• Gel selection:

  • Use 12% SDS-PAGE gels for optimal resolution of 38-60 kDa range proteins

• Antibody selection:

  • Primary antibody concentration: typically 1:1000-1:2000 dilution

  • Use antibodies targeting conserved epitopes to detect all forms

• Controls:

  • Include wild-type CLN5 expressing cells as positive controls

  • Consider using CLN5 mutants (e.g., glycosylation site mutants) as comparative controls

This approach allows detection of both the mature glycosylated forms (~60 kDa) and deglycosylated forms (~38 kDa) of the protein, providing insights into CLN5 processing and maturation.

How can I use CLN5 antibodies to investigate trafficking defects caused by disease-associated mutations?

CLN5 mutations frequently disrupt proper protein trafficking to lysosomes. A methodological approach to investigate trafficking defects includes:

• Transient expression system setup:

  • Transfect cells with wild-type and mutant CLN5 constructs (with epitope tags if needed)

  • Allow 24-48 hours for expression

• Translation inhibition approach:

  • Treat a subset of transfected cells with cycloheximide (50μM for 5 hours)

  • This allows tracking of existing protein while preventing new synthesis

• Co-localization analysis:

  • Perform immunofluorescence with the CLN5 antibody

  • Co-stain with lysosomal markers (e.g., LAMP-1)

  • Use confocal microscopy for high-resolution imaging

• Quantitative assessment:

  • Calculate Pearson's correlation coefficient between CLN5 and LAMP-1 signals

  • Compare wild-type and mutant co-localization percentages

This methodology has revealed that disease-causing mutations like p.Arg145Pro significantly impair lysosomal trafficking, with mutant proteins remaining in the ER while wild-type CLN5 properly localizes to lysosomes after cycloheximide treatment .

What considerations should be made when using CLN5 antibodies to study protein-protein interactions?

When investigating CLN5 interactions with other proteins, consider:

• Antibody epitope location:

  • Ensure the antibody epitope doesn't overlap with interaction domains

  • C-terminal targeted antibodies may be preferable as N-terminal processing occurs

• Experimental approaches:

  • Immunoprecipitation: Use antibodies conjugated to solid supports

  • Proximity ligation assays: Combine CLN5 antibodies with antibodies against potential interactors

  • FRET/BRET: For live-cell interaction studies (requires fluorescent protein tagging)

• Control considerations:

  • Use IgG controls to assess non-specific binding

  • Include known CLN5 interactors as positive controls

  • Consider using CLN5-deficient cells as negative controls

CLN5 has been reported to interact with other NCL proteins and lysosomal proteins, making these interactions important for understanding disease mechanisms . The antibody selection should be optimized based on the specific interaction being studied.

What are the optimal fixation and permeabilization conditions for CLN5 immunofluorescence staining?

For optimal immunofluorescence detection of CLN5:

• Fixation:

  • Use 4% paraformaldehyde for 15-20 minutes at room temperature

  • Alternative: methanol fixation (100%, -20°C, 10 minutes) for membrane protein preservation

• Permeabilization:

  • For paraformaldehyde-fixed cells: 0.1-0.2% Triton X-100 for 10 minutes

  • For methanol-fixed cells: additional permeabilization is typically unnecessary

• Blocking:

  • 3-5% BSA in PBS or 5-10% serum (from secondary antibody host species)

  • Include 0.1% Tween-20 to reduce background

• Antibody incubation:

  • Primary: Overnight at 4°C or 1-2 hours at room temperature

  • Secondary: 1 hour at room temperature

• Washing:

  • Multiple PBS washes (3-5 times, 5 minutes each) between steps

For co-localization with lysosomal markers, these conditions have been successfully used to demonstrate mutant CLN5 trafficking defects, showing reduced co-localization with LAMP-1 compared to wild-type CLN5 .

How can I evaluate the specificity of a CLN5 antibody in my experimental system?

Ensuring antibody specificity is critical for reliable results. Methodological approaches include:

• Genetic validation:

  • Use CLN5 knockout/knockdown cells as negative controls

  • Utilize overexpression systems with tagged CLN5 for positive controls

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare staining/blotting patterns with and without peptide blocking

• Multiple antibody approach:

  • Compare staining patterns with antibodies targeting different CLN5 epitopes

  • Consistent results across antibodies increase confidence in specificity

• Western blot validation:

  • Verify detection of expected molecular weight bands (38-60 kDa range)

  • Compare pattern with published literature on CLN5 expression

• Deglycosylation analysis:

  • Treat samples with EndoH and PNGaseF to confirm expected shift to ~38 kDa

This systematic approach helps ensure that signals detected by the CLN5 antibody represent authentic target protein rather than cross-reactive artifacts.

How should I design experiments to study CLN5 glycosylation patterns across different cell types?

CLN5 glycosylation varies between cell types, requiring careful experimental design:

• Cell selection:

  • Include neuronal and non-neuronal cell lines

  • Consider patient-derived fibroblasts or iPSCs

  • Include species-relevant cells if studying across species

• Expression systems:

  • Compare endogenous versus overexpressed CLN5

  • For overexpression, use physiologically relevant promoters when possible

• Glycosylation analysis protocol:

  Step	Method	Expected Outcome
  Baseline characterization	Western blot with CLN5 antibody	Multiple bands (40-60 kDa)
  EndoH treatment	Overnight incubation with EndoH	Removal of high-mannose glycans
  PNGaseF treatment	Overnight incubation with PNGaseF	Complete deglycosylation to ~38 kDa
  Tunicamycin treatment	Pretreatment of live cells	Prevention of N-glycosylation

• Data analysis:

  • Quantify band intensity ratios between glycosylated and deglycosylated forms

  • Compare glycosylation patterns across cell types and conditions

This approach can reveal tissue-specific post-translational processing of CLN5, which may contribute to differential pathology in CLN5 disease variants.

What controls should be included when studying trafficking defects of CLN5 disease mutants?

When investigating CLN5 trafficking:

• Essential controls:

  • Wild-type CLN5 expression (positive control)

  • Empty vector transfection (negative control)

  • Known trafficking mutants (e.g., p.Y392X Finnish founder mutation)

• Treatment controls:

  • Untreated baseline samples

  • Cycloheximide-treated samples to track protein trafficking

  • Brefeldin A treatment to block ER-to-Golgi transport

• Co-localization markers:

  • ER markers (e.g., calnexin, PDI)

  • Golgi markers (e.g., GM130)

  • Lysosomal markers (e.g., LAMP-1, LAMP-2)

• Time course analysis:

  • Evaluate trafficking at multiple time points (6, 12, 24, 48 hours)

  • Pulse-chase labeling for protein tracking

Such comprehensive controls enable accurate assessment of mutant protein trafficking dynamics, as demonstrated in studies showing that mutations like p.Arg145Pro disrupt lysosomal targeting compared to wild-type CLN5 .

Why might I observe different molecular weight bands than expected when using CLN5 antibodies in Western blots?

Discrepancies in CLN5 molecular weight can arise from several factors:

• Post-translational modifications:

  • Variable glycosylation patterns between cell types

  • Differential N-terminal processing of CLN5

  • Potential ubiquitination or other modifications

• Technical variables:

  • Sample preparation methods (denaturing vs. non-denaturing)

  • Gel percentage affecting protein migration

  • Insufficient denaturation or reduction

• Antibody specificity:

  • Epitope location affecting detection of processed forms

  • Potential cross-reactivity with related proteins

• Biological variables:

  Variable	Potential Impact	Resolution
  Cell type differences	Altered glycosylation patterns	Compare with deglycosylated samples
  Alternative translation start sites	Multiple protein isoforms (47, 44, 41, 39 kDa)	Use N- and C-terminal antibodies for verification
  Protein degradation	Appearance of lower MW bands	Include protease inhibitors during preparation
  Disease mutations	Altered processing or stability	Compare with wild-type controls

If unexpected bands are observed, verification through deglycosylation analysis and comparison with published literature on CLN5 expression patterns is recommended .

How can I differentiate between nonspecific staining and genuine CLN5 localization in immunofluorescence experiments?

To distinguish specific from nonspecific CLN5 staining:

• Critical controls:

  • CLN5-deficient cells or tissues (genetic negative control)

  • Isotype-matched IgG control (antibody negative control)

  • Peptide competition (epitope blocking control)

• Validation approaches:

  • Multiple antibody validation using different CLN5 epitopes

  • Correlation with tagged CLN5 expression

  • siRNA knockdown showing signal reduction

• Co-localization analysis:

  • Expected: CLN5 should primarily co-localize with lysosomal markers in wild-type cells

  • ER co-localization may indicate immature or mutant protein

• Signal characteristics:

  • Specific staining: Punctate, primarily perinuclear pattern in wild-type cells

  • Nonspecific staining: Often diffuse, present in negative controls, or unusually intense

Studies have demonstrated that wild-type CLN5 shows punctate staining with increased lysosomal co-localization after cycloheximide treatment, while mutants like p.Arg145Pro show disrupted trafficking patterns . These established patterns provide reference points for evaluating staining specificity.

How can CLN5 antibodies be used to study autophagy dysregulation in NCL models?

CLN5 deficiency has been linked to autophagy disruption. Methodological approaches include:

• Autophagy marker analysis:

  • Use CLN5 antibodies alongside autophagy markers (LC3-I/II, p62/SQSTM1)

  • Compare marker expression between wild-type and CLN5-mutant cells

• Autophagy flux assessment:

  • Treat cells with autophagy modulators (bafilomycin A1, rapamycin)

  • Monitor changes in LC3-II accumulation with/without CLN5 function

• Co-localization experiments:

  • Assess CLN5 localization relative to autophagosomes/autolysosomes

  • Monitor lysosomal function via LysoTracker or pH-sensitive probes

• Live cell imaging:

  • Use fluorescently-tagged CLN5 together with autophagy reporters

  • Perform time-lapse imaging to track dynamic interactions

• Electron microscopy:

  • Immunogold labeling with CLN5 antibodies

  • Ultrastructural analysis of autophagosome/autolysosome morphology

Research has shown that CLN5 mutations can affect the expression of autophagy-related proteins like LC3I/II, LAMP-1, and p62/SQSTM1 , suggesting CLN5's involvement in autophagy regulation.

What methodologies can I use to study the recently discovered BMP synthase activity of CLN5?

The bis(monoacylglycero)phosphate (BMP) synthase activity of CLN5 represents a recent discovery that can be investigated through:

• In vitro enzymatic assays:

  • Immunoprecipitate CLN5 using specific antibodies

  • Assess BMP synthesis using purified lysophosphatidylglycerol substrates

  • Measure product formation via mass spectrometry

• Substrate preference analysis:

  • Test CLN5 activity with various LPGs (LPG 14:0, LPG 16:0, LPG 18:0, LPG 18:1)

  • Quantify relative activity to determine chain length preferences

• Structural impact of mutations:

  • Compare wild-type and mutant CLN5 enzymatic activity

  • Correlate structure predictions with functional outcomes

• Cellular BMP dynamics:

  • Use CLN5 antibodies alongside lipidomic analysis

  • Compare BMP levels in control vs. CLN5-deficient cells

CLN5 has demonstrated BMP synthase activity through transacylation of lysophosphatidylglycerol molecules, with preference for longer chain lengths . This enzymatic function provides a new direction for understanding CLN5's role in lysosomal function and intracellular cholesterol homeostasis.

How might single-cell analysis with CLN5 antibodies advance our understanding of cell-type specific pathology in NCL?

Single-cell approaches using CLN5 antibodies could provide unprecedented insights:

• Single-cell immunophenotyping:

  • Combine CLN5 antibodies with cell-type specific markers

  • Use flow cytometry or mass cytometry (CyTOF) for quantitative analysis

  • Identify differential expression across neural cell populations

• Spatial transcriptomics integration:

  • Correlate CLN5 protein localization with gene expression patterns

  • Map cell-type specific vulnerability in NCL pathology

• Patient-derived organoid applications:

  • Apply CLN5 antibodies to 3D brain organoids from patient iPSCs

  • Compare wild-type and mutant organoid development

• Quantitative approach:

  Analysis Level	Technique	Expected Insight
  Protein expression	Single-cell Western	Cell-to-cell variation in CLN5 levels
  Subcellular localization	Super-resolution microscopy	Nanoscale distribution in different cell types
  Interaction networks	Proximity labeling + proteomics	Cell-type specific interactome

Since CLN5 shows both neuronal and glial expression in the brain , this approach could reveal why certain cell populations are more vulnerable to CLN5 dysfunction than others, potentially identifying cell-specific therapeutic targets.

What methodological approaches can determine if CLN5 antibodies detect the recently proposed palmitoyl protein thioesterase activity?

CLN5 has been suggested to possess palmitoyl protein thioesterase (S-depalmitoylation) activity . To investigate this function:

• Activity assays:

  • Immunoprecipitate CLN5 using specific antibodies

  • Assess thioesterase activity with fluorogenic substrates

  • Compare activity between wild-type and mutant proteins

• Substrate identification:

  • Perform acyl-biotin exchange (ABE) assays

  • Compare palmitoylated proteome between control and CLN5-deficient cells

  • Validate potential substrates through direct interaction studies

• Structural biology approach:

  • Model CLN5 catalytic domain based on known thioesterases

  • Design site-directed mutagenesis to target predicted catalytic residues

  • Correlate structural predictions with functional outcomes

• Comparative analysis:

  • Compare CLN5 activity with established depalmitoylases (e.g., PPT1/CLN1)

  • Investigate potential functional redundancy or specificity

This methodological framework could help clarify whether the proposed S-depalmitoylation activity represents a primary physiological function of CLN5 or a secondary activity, providing crucial insights into CLN5 biology and NCL pathogenesis.

What are the recommended best practices for long-term storage and handling of CLN5 antibodies to maintain optimal performance?

For optimal antibody performance and longevity:

• Storage recommendations:

  • Store antibody aliquots at -20°C to -80°C for long-term stability

  • Avoid repeated freeze-thaw cycles (make small working aliquots)

  • For HRP-conjugated antibodies, add 50% glycerol for freeze protection

• Working solution handling:

  • Store working dilutions at 4°C for up to 1 month

  • Add preservatives (0.02% sodium azide) for longer storage

  • Protect HRP-conjugated antibodies from direct light exposure

• Quality control measures:

  • Periodically test antibody performance against positive controls

  • Include antibody validation steps in each experimental series

  • Document lot numbers and performance characteristics

• Optimization recommendations:

  Application	Dilution Range	Buffer Recommendation
  Western blot	1:1000-1:5000	TBST with 3-5% BSA
  IHC-P	1:100-1:500	PBS with 1% BSA
  ICC/IF	1:200-1:1000	PBS with 1% BSA

Following these guidelines will help ensure consistent results with CLN5 antibodies across multiple experiments and over extended research timelines.

How should I approach the validation of a new lot of CLN5 antibody for cross-lot consistency?

When validating a new antibody lot:

• Side-by-side comparison:

  • Run parallel experiments with old and new lots

  • Use identical samples, conditions, and protocols

• Comprehensive validation panel:

  • Test across multiple applications (WB, IHC, IF)

  • Include positive controls (cells known to express CLN5)

  • Include negative controls (CLN5-deficient samples if available)

• Quantitative assessment:

  • Compare signal-to-noise ratios between lots

  • Assess staining intensity and pattern consistency

  • Evaluate detection sensitivity with dilution series

• Epitope verification:

  • Consider peptide competition assays to confirm epitope recognition

  • Verify detection of known CLN5 forms (glycosylated/deglycosylated)

• Documentation:

  • Maintain detailed records of lot performance characteristics

  • Document any adjustments needed for optimal results with new lot