NHLRC1 Antibody, FITC conjugated

What is NHLRC1 and why is it significant in neurological research?

NHLRC1 (NHL repeat-containing protein 1), also known as Malin or EPM2B, is a single subunit E3 ubiquitin ligase that contains a RING-type zinc finger domain and six NHL-repeat domains . This protein is of significant research interest because:

• It forms a functional complex with laforin (encoded by EPM2A) to regulate glycogen synthesis

• It suppresses cellular toxicity of misfolded proteins by promoting their degradation through the ubiquitin-proteasome system (UPS)

• Mutations in NHLRC1 are causative for Lafora disease (progressive myoclonic epilepsy type 2), a fatal autosomal recessive neurodegenerative disorder

The study of NHLRC1 provides critical insights into protein quality control mechanisms and the pathogenesis of neurodegenerative diseases characterized by protein aggregation.

How does FITC conjugation to NHLRC1 antibodies occur at the molecular level?

FITC (Fluorescein Isothiocyanate) conjugation to NHLRC1 antibodies involves a chemical reaction between the isothiocyanate group of FITC and primary amines (typically lysine residues) on the antibody. The process follows these steps:

• The reactive fluorescein molecule (FITC) is dissolved in an organic solvent like DMSO immediately before use due to its instability

• The antibody is prepared in a carbonate or borate buffer at pH 8.0-9.5 to ensure that lysine ε-amino groups are deprotonated for optimal reactivity

• The FITC solution is slowly added to the antibody solution while gently mixing to achieve the desired FITC:antibody molar ratio (typically aiming for 3-6 FITC molecules per antibody)

• The reaction proceeds at room temperature in the dark for 1-2 hours

• Unconjugated FITC is removed by gel filtration or dialysis against phosphate-buffered saline (PBS)

This conjugation technique creates a stable thiourea bond between FITC and the antibody, enabling fluorescent detection of NHLRC1 while maintaining the antibody's binding specificity.

What are the optimal storage conditions for maintaining FITC-conjugated NHLRC1 antibody stability?

For maximum stability and performance of FITC-conjugated NHLRC1 antibodies:

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

• Keep in storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Protect from continuous light exposure, which can gradually reduce fluorescence intensity

• Maintain in the dark during all storage and handling steps

• Avoid contamination by using sterile technique during aliquoting

Some commercial FITC-conjugated antibody preparations remain stable for up to one year when properly stored at -20°C . When preparing to use the antibody, thaw a single aliquot on ice and keep protected from light until use in the experimental procedure.

What are the recommended dilutions and applications for FITC-conjugated NHLRC1 antibodies?

FITC-conjugated NHLRC1 antibodies can be utilized across several applications with the following recommended dilutions:

For NHLRC1 detection in neuronal tissues or cells expressing NHLRC1 at low levels, starting with more concentrated antibody dilutions (1:20-1:50) is advisable. For all applications, include proper controls and perform titration experiments to determine the optimal signal-to-noise ratio in your specific experimental system .

How can I optimize an immunofluorescence protocol specifically for detecting NHLRC1 using FITC-conjugated antibodies?

An optimized immunofluorescence protocol for NHLRC1 detection with FITC-conjugated antibodies requires careful attention to several parameters:

• Cell preparation and fixation:

  • Grow cells on chamber slides or coverslips to 70-80% confluence

  • Fix with 4% paraformaldehyde for 20 minutes at room temperature

  • Permeabilize with 0.5% Triton X-100 for 5 minutes

• Blocking and antibody incubation:

  • Block with PBS containing 10% fetal bovine serum for 20-30 minutes to reduce non-specific binding

  • Dilute FITC-conjugated NHLRC1 antibody (1:50-1:200) in blocking solution

  • Incubate cells with antibody solution for 1 hour at room temperature in the dark

• Washing and mounting:

  • Wash cells 2-3 times for 5 minutes each with PBS to remove unbound antibody

  • Mount with anti-fade mounting medium containing DAPI for nuclear counterstaining

  • Seal edges with nail polish to prevent drying

• Visualization:

  • Observe using a fluorescence microscope equipped with appropriate FITC filter set (excitation ~490 nm, emission ~525 nm)

  • Capture images using exposure settings that avoid photobleaching

For improved specificity when studying NHLRC1's interaction with the ubiquitin-proteasome system, co-staining with antibodies against known interaction partners like laforin (EPM2A) using different fluorophores (e.g., Texas Red) can provide valuable colocalization data .

Why might I observe high background or non-specific staining when using FITC-conjugated NHLRC1 antibodies?

High background or non-specific staining with FITC-conjugated NHLRC1 antibodies can result from several factors:

Research has shown that FITC-conjugated antibodies with higher labeling indices tend to be more sensitive but are also more prone to non-specific staining . For NHLRC1 detection specifically, the optimal FITC:antibody ratio should be determined empirically by comparing several conjugates with different labeling densities in your experimental system.

How can I verify the specificity of my FITC-conjugated NHLRC1 antibody?

To confirm the specificity of your FITC-conjugated NHLRC1 antibody, implement these validation approaches:

• Positive and negative control samples:

  • Use cell lines with known NHLRC1 expression (e.g., L02 cells)

  • Include cells where NHLRC1 is absent or knocked down (siRNA)

  • Examine tissues with differential NHLRC1 expression (e.g., brain versus non-neural tissues)

• Competitive inhibition:

  • Pre-incubate the antibody with excess recombinant NHLRC1 protein before staining

  • Signal should be significantly reduced or eliminated if the antibody is specific

• Dual validation with unconjugated antibody:

  • Compare staining pattern between FITC-conjugated and unconjugated NHLRC1 antibody

  • Use Western blot analysis to verify the detection of a single band at the expected molecular weight (42-47 kDa)

• Epitope mapping validation:

  • For NHLRC1 specifically, confirm recognition of the correct domain (RING domain or NHL repeats)

  • Check reactivity against recombinant fragments representing different domains of NHLRC1

• Cross-validation with alternate detection methods:

  • Compare immunofluorescence results with immunohistochemistry or Western blot data

  • Use multiple antibodies targeting different epitopes of NHLRC1

Remember that proper validation should demonstrate not only presence of signal in positive samples but also appropriate subcellular localization (NHLRC1 localizes primarily to the endoplasmic reticulum and to a lesser extent in the nucleus) .

How does FITC conjugation affect the binding kinetics and sensitivity of NHLRC1 antibody detection?

FITC conjugation can significantly impact NHLRC1 antibody performance through several mechanisms:

• Effect on binding affinity:

  • Research demonstrates a negative correlation between FITC-labeling index and binding affinity for target antigens

  • Each FITC molecule conjugated to lysine residues alters the antibody's charge profile and potentially its conformational stability

  • Over-labeling (>6 FITC molecules per antibody) can substantially reduce binding affinity by modifying lysines in or near the antigen-binding region

• Sensitivity considerations:

  • Moderately labeled antibodies (3-4 FITC molecules per antibody) often provide optimal balance between brightness and binding affinity

  • Higher labeling ratios increase fluorescence intensity but may compromise specificity and increase background

  • For detecting low-abundance NHLRC1 expression, signal amplification methods may be preferable to excessive FITC labeling

• Quantitative implications:

  • For quantitative applications (e.g., measuring NHLRC1 expression levels), consistent FITC:antibody ratios between batches is critical

  • Standardize using fluorescent beads or calibration standards to normalize between experiments

  • Consider using secondary antibody detection systems when precise quantitation is required

When selecting FITC-conjugated NHLRC1 antibodies for research, evaluate technical documentation regarding the FITC:protein ratio and validate performance empirically in your experimental system. For detecting subtle changes in NHLRC1 expression or localization, antibodies with moderate FITC labeling typically provide the best balance of sensitivity and specificity .

What methodological considerations are important when studying NHLRC1 mutations using FITC-conjugated antibodies?

When investigating NHLRC1 mutations associated with Lafora disease using FITC-conjugated antibodies, researchers should consider several methodological factors:

• Epitope accessibility in mutated proteins:

  • Confirm that your FITC-conjugated antibody's epitope is not directly affected by the mutation of interest

  • NHLRC1 mutations in the RING domain (C46Y, P69A) or NHL domains (D146N, L261P) may alter protein conformation and epitope accessibility

  • Use antibodies targeting regions distant from mutation sites or multiple antibodies recognizing different epitopes

• Expression level variations:

  • Mutations may affect NHLRC1 protein stability or expression levels

  • Standardize detection protocols with appropriate loading controls

  • Consider complementary techniques like Western blotting to quantify expression differences

• Subcellular localization changes:

  • NHLRC1 mutations can alter subcellular trafficking and localization

  • Use confocal microscopy with z-stack imaging to accurately determine 3D localization patterns

  • Co-stain with organelle markers (ER, nucleus) to detect mislocalization of mutant proteins

• Functional interaction studies:

  • For studying how mutations affect NHLRC1's interaction with binding partners like laforin:

    • Perform co-immunoprecipitation followed by immunofluorescence

    • Use dual-color labeling with FITC-conjugated NHLRC1 antibody and Texas Red-conjugated antibodies against interaction partners

    • Employ proximity ligation assays for higher sensitivity in detecting protein-protein interactions

• Analysis of polyglucosan inclusions:

  • When studying Lafora bodies, combine FITC-conjugated NHLRC1 antibody staining with periodic acid-Schiff (PAS) staining

  • Optimize fixation and permeabilization to preserve both protein epitopes and glycogen-like aggregates

For researching specific NHLRC1 mutations like C46Y, P69A, D146N and L261P, cell models expressing these variants should be carefully validated to ensure that the mutant proteins are recognized by your FITC-conjugated antibody with similar efficiency as the wild-type protein .

How can FITC-conjugated NHLRC1 antibodies be effectively employed in co-localization studies with other proteins in the ubiquitin-proteasome pathway?

To effectively use FITC-conjugated NHLRC1 antibodies in co-localization studies with ubiquitin-proteasome pathway components:

• Multi-channel fluorescence imaging setup:

  • Pair FITC (green fluorescence, Ex/Em: 495/519 nm) with spectrally distinct fluorophores like Texas Red or Cy3 (red) and Cy5 (far-red) for multi-protein detection

  • Ensure your microscope has appropriate filter sets to minimize spectral overlap

  • For complex co-localization studies, use confocal microscopy with sequential scanning to prevent bleed-through

• Sample preparation for optimal co-localization analysis:

  • Fix cells using paraformaldehyde (4%) to preserve protein interactions and spatial relationships

  • For membrane proteins, avoid harsh permeabilization; use 0.1-0.2% Triton X-100 or 0.1% saponin

  • For co-staining of NHLRC1 with ubiquitin or proteasome components:

    • FITC-conjugated NHLRC1 antibody (1:100)

    • Texas Red-conjugated anti-ubiquitin or anti-proteasome antibodies (1:200)

• Validated co-localization protocols for NHLRC1 interactions:

  • For NHLRC1-laforin interactions:

    • Co-stain with FITC-conjugated NHLRC1 and Texas Red-conjugated laforin antibodies

    • Focus on areas with Lafora body formation in neuronal models

  • For NHLRC1-Dvl2 (Wnt signaling) co-localization:

    • Use FITC-conjugated anti-ubiquitin antibody together with Texas Red-conjugated anti-Dvl2 in cells expressing NHLRC1

    • Examine localization changes upon proteasome inhibition (e.g., MG132 treatment)

• Quantitative co-localization analysis:

  • Calculate Pearson's correlation coefficient or Manders' overlap coefficient

  • Use JACoP plugin in ImageJ or similar software for unbiased analysis

  • Control for random co-localization using protein pairs known not to interact

• Confirming functional associations:

  • Complement imaging with biochemical approaches (co-immunoprecipitation, proximity ligation)

  • For NHLRC1's E3 ligase activity assessment, monitor ubiquitination status of target proteins in parallel with co-localization studies

When studying NHLRC1's role in targeting proteins for degradation, time-course experiments combining FITC-conjugated NHLRC1 antibody with proteasome markers can reveal dynamic changes in co-localization during the protein degradation process .

What are the critical experimental controls needed when using FITC-conjugated NHLRC1 antibodies for detecting Lafora bodies in clinical samples?

When using FITC-conjugated NHLRC1 antibodies to detect Lafora bodies in clinical samples, implement these essential controls:

• Antibody specificity controls:

  • Isotype control: Use FITC-conjugated non-specific immunoglobulin (same isotype as NHLRC1 antibody)

  • Absorption control: Pre-incubate FITC-conjugated NHLRC1 antibody with excess recombinant NHLRC1 protein

  • Secondary antibody-only control (if using indirect detection method)

• Tissue-specific controls:

  • Positive control: Include known Lafora disease tissue sections with confirmed Lafora bodies

  • Negative control: Include age-matched normal tissue sections

  • For brain tissue: Use antigen retrieval with TE buffer pH 9.0 (or citrate buffer pH 6.0 as alternative)

• Autofluorescence mitigation:

  • Include unstained tissue section to assess natural autofluorescence

  • Use Sudan Black B (0.1-0.3%) treatment to quench tissue autofluorescence

  • Employ spectral unmixing if confocal microscope with spectral detection is available

• Validation with conventional staining:

  • Perform parallel PAS (Periodic Acid-Schiff) staining on adjacent sections

  • Compare FITC-NHLRC1 immunolabeling with PAS-positive inclusions

  • For definitive confirmation, consider dual PAS-immunofluorescence labeling

• Inter-laboratory standardization:

  • Document acquisition parameters (exposure time, gain, filter settings)

  • Include fluorescent intensity calibration standards

  • Process and image reference samples alongside test samples

For clinical diagnosis of Lafora disease, immunofluorescence with FITC-conjugated NHLRC1 antibodies should be used as a complementary technique to genetic testing for NHLRC1 mutations, as some mutations may affect antibody recognition or protein expression levels . Lafora bodies should show positive staining for NHLRC1 and typically appear as cytoplasmic inclusions in neurons, but can also be found in liver, muscle, and heart tissue samples .

How can FITC-conjugated NHLRC1 antibodies be utilized in studying the relationship between NHLRC1 and glycogen metabolism disorders?

FITC-conjugated NHLRC1 antibodies offer valuable tools for investigating NHLRC1's role in glycogen metabolism and related disorders:

• Visualization of glycogen-associated protein complexes:

  • Use FITC-conjugated NHLRC1 antibodies (1:50-1:100) alongside antibodies against:

    • Laforin (EPM2A) - NHLRC1's functional partner in glycogen regulation

    • R5/PTG - regulatory subunit of protein phosphatase 1 involved in glycogen synthesis

    • Glycogen synthase (GS) - key enzyme in glycogen formation

  • Perform z-stack confocal imaging to visualize 3D relationships between these proteins and glycogen particles

• Characterization of polyglucosan bodies:

  • Combine FITC-NHLRC1 immunofluorescence with glycogen staining methods:

    • Periodic Acid-Schiff (PAS) staining modified for fluorescence microscopy

    • Specific glycogen-binding protein probes

  • This dual approach allows correlation between NHLRC1 localization and abnormal glycogen structures

• Monitoring dynamic changes in glycogen metabolism:

  • Design time-course experiments using cell models with fluorescently-tagged NHLRC1 and glycogen metabolism enzymes

  • Apply glucose challenges or glycogen metabolism inhibitors to observe redistribution of NHLRC1 relative to glycogen particles

  • Quantify changes in co-localization coefficients over time

• Analyzing mutation-specific effects on glycogen regulation:

  • Compare wild-type and mutant NHLRC1 localization relative to glycogen accumulation

  • Data from Lafora disease patients showed that mutations (C46Y, P69A, D146N, and L261P) prevent the laforin-malin complex from downregulating R5/PTG-induced glycogen synthesis

  • Quantify differences in glycogen levels and NHLRC1 association across mutations using fluorescence intensity measurements

• Cross-species comparison in model systems:

  • Validate FITC-conjugated NHLRC1 antibody reactivity across relevant animal models

  • Compare glycogen metabolism phenotypes and NHLRC1 distribution in mouse, rat, and human samples

  • Document species-specific differences in NHLRC1 association with glycogen particles

These approaches can help elucidate the molecular mechanisms through which NHLRC1 mutations lead to the abnormal glycogen accumulation characteristic of Lafora disease, potentially identifying targets for therapeutic intervention .

What considerations are important when using FITC-conjugated NHLRC1 antibodies for high-resolution microscopy techniques?

When employing FITC-conjugated NHLRC1 antibodies for advanced microscopy techniques, researchers should address these technical considerations:

• Super-resolution microscopy optimization:

  • For STED (Stimulated Emission Depletion) microscopy:

    • FITC is not ideal due to photobleaching; consider converting to more photostable fluorophores like Alexa Fluor 488

    • Use mounting media with anti-fade agents formulated for super-resolution

    • Optimize depletion laser power to balance resolution and signal preservation

  • For STORM/PALM techniques:

    • FITC's photoswitching properties are suboptimal; consider immunolabeling with primary anti-NHLRC1 followed by secondary antibodies conjugated to superior photoswitching dyes

    • Use oxygen-scavenging buffer systems to enhance photostability

    • Ensure high labeling density for accurate reconstruction

• Sample preparation refinements:

  • For optimal NHLRC1 visualization at nanoscale resolution:

    • Use thinner sections (70-100 nm) for improved signal-to-noise ratio

    • Apply gentler fixation protocols (2% PFA instead of 4%) to preserve epitope accessibility

    • Consider membrane extraction techniques to better visualize association with cytoskeletal elements and organelles

• Multi-color high-resolution imaging approaches:

  • When combining FITC-conjugated NHLRC1 with other fluorophores:

    • Select fluorophores with minimal spectral overlap

    • For 3-4 color imaging, use sequential acquisition with careful channel alignment

    • Consider chromatic aberration correction for accurate co-localization analysis at nanoscale resolution

• Quantitative aspects for nanoscale analysis:

  • For accurate quantification of NHLRC1 distribution and clustering:

    • Use appropriate density-based clustering algorithms

    • Apply calibration standards to account for different detection efficiencies between fluorophores

    • Consider molecular counting approaches to estimate absolute numbers of NHLRC1 molecules in complexes

• Live-cell super-resolution considerations:

  • For studying dynamic behavior of NHLRC1:

    • FITC photobleaching limits extended imaging; consider generating cells expressing NHLRC1 fused to more photostable fluorescent proteins

    • Use reduced illumination intensity with longer acquisition times

    • Apply denoising algorithms optimized for low-signal conditions

By addressing these technical aspects, researchers can leverage high-resolution microscopy to elucidate previously unresolvable details of NHLRC1's interaction with binding partners and its role in protein quality control pathways at the nanoscale level.