ch25hl2 Antibody

What is CH25H/ch25hl2 and what is its primary function in cellular metabolism?

CH25H (cholesterol 25-hydroxylase) is a protein that catalyzes the formation of 25-hydroxycholesterol from cholesterol. In humans, this approximately 31.7 kilodalton protein is also known as C25H, cholesterol 25-monooxygenase, and h25OH . The zebrafish orthologs include ch25hl1.1, ch25hl1.2, and ch25hl2 .

Its primary functions include:

• Repressing cholesterol biosynthetic enzymes through 25-hydroxycholesterol production

• Regulating cell positioning and movement in lymphoid tissues

• Exhibiting broad antiviral activities against enveloped viruses

• Playing roles in Leydig cell differentiation in the testis

• Restraining inflammation in macrophages by preventing cholesterol overload

The enzyme prevents mitochondrial DNA release and subsequent activation of the AIM2 inflammasome, which is crucial for maintaining cellular homeostasis in response to cholesterol flux.

How can researchers validate the specificity of CH25H/ch25hl2 antibodies?

Antibody validation requires multiple complementary approaches:

• Western blot analysis with positive and negative controls:

  • Use tissues known to express (e.g., macrophages) and not express the target

  • Include knockout/knockdown samples where available

  • Verify molecular weight (approximately 31.7 kDa for human CH25H)

• Immunohistochemical validation:

  • Compare staining patterns with published literature

  • Perform antigen blocking experiments

  • Use multiple antibodies targeting different epitopes

• Immunofluorescence crosschecking:

  • Co-localization with cellular markers associated with CH25H expression

  • RNA in situ hybridization correlation

• Recombinant protein controls:

  • Test antibody against purified recombinant CH25H protein

  • Perform dose-response analyses

• Cross-reactivity assessment:

  • Test against related family members to ensure specificity

  • Particularly important when studying zebrafish orthologs (ch25hl1.1, ch25hl1.2, ch25hl2)

What are the common applications of CH25H antibodies in research?

Most CH25H antibodies are tested and validated for Western blot (WB), ELISA, and immunohistochemistry applications, with variable cross-reactivity across species including human, mouse, rat and zebrafish .

How does CH25H/ch25hl2 contribute to antiviral immunity mechanisms?

CH25H plays a significant role in innate immunity against enveloped viruses through multiple mechanisms:

• 25-hydroxycholesterol production: As an interferon-stimulated gene, CH25H produces 25-hydroxycholesterol which has broad antiviral activities against various enveloped viruses including vesicular stomatitis virus (VSV) and SARS-CoV-2 .

• Membrane modification: 25-hydroxycholesterol activates ER-localized ACAT enzyme, inducing internalization of accessible cholesterol from the plasma membrane. This membrane modification restricts viral fusion, particularly for SARS-CoV-2 S protein-mediated fusion .

• Viral escape mechanisms: The selective pressure exerted by antibodies against viral proteins (such as SARS-CoV-2 spike) can lead to escape mutations. Understanding how CH25H influences membrane composition can provide insights into viral adaptation strategies .

For studying these mechanisms, researchers should consider combining:

• Transcriptional analysis to measure CH25H induction following interferon stimulation

• Lipidomic profiling to quantify oxysterol production

• Viral entry assays with and without CH25H expression

• Membrane fluidity assessments to determine cholesterol redistribution effects

What approaches can be used to develop single-domain antibodies (nanobodies) against CH25H/ch25hl2?

Nanobody development against CH25H could follow protocols similar to those used for other antigens:

• Camelid immunization:

  • Llamas or alpacas are commonly used for nanobody generation

  • Immunization with purified CH25H protein or specific domains

  • Multiple booster shots over 6-8 weeks

• Library construction and screening:

  • Blood collection to isolate peripheral blood lymphocytes

  • VHH (variable domain of heavy chain antibodies) gene amplification

  • Phage display or yeast display library construction

  • Selection against immobilized CH25H protein

• Validation and engineering:

  • Sequence analysis of enriched clones

  • Expression in bacterial or mammalian systems

  • Affinity maturation if required

  • Biophysical characterization (thermal stability, aggregation propensity)

• Functional testing:

  • Enzyme inhibition assays

  • Cellular localization studies

  • In vitro and in vivo imaging applications

Nanobodies against CH25H could offer advantages for studying this enzyme in living cells due to their small size (~15 kDa), high stability, and ability to access epitopes that are inaccessible to conventional antibodies .

How can researchers resolve contradictory data on CH25H expression patterns between different antibody-based detection methods?

When faced with contradictory CH25H expression data, consider these methodological approaches:

• Multi-epitope antibody strategy:

  • Use antibodies targeting different regions of CH25H

  • Compare monoclonal and polyclonal antibodies

  • Assess epitope accessibility under different experimental conditions

• Complementary non-antibody techniques:

  • RT-qPCR for mRNA expression correlation

  • RNA-sequencing data analysis

  • CRISPR-tagged endogenous protein visualization

• Assay-specific optimizations:

  • For Western blot: Compare different lysis buffers, reducing agents, and denaturation conditions

  • For IHC/IF: Test multiple fixation and antigen retrieval methods

  • For flow cytometry: Compare intracellular staining protocols

• Biological variables control:

  • Ensure identical cell/tissue sources

  • Control for activation state (CH25H is induced by interferons)

  • Consider post-translational modifications affecting epitope accessibility

• Quantitative comparison:

  • Establish standard curves with recombinant protein

  • Use multiple reference proteins/genes for normalization

  • Perform spike-in controls to assess recovery efficiency

What protocols yield optimal results for immunoprecipitation using CH25H antibodies?

Successful immunoprecipitation of CH25H requires careful protocol optimization:

• Cell lysis optimization:

  • Use mild detergents (0.5-1% NP-40, CHAPS, or digitonin)

  • Include protease inhibitors and phosphatase inhibitors

  • Maintain cold temperature throughout the procedure

  • Consider membrane fractionation, as CH25H is a membrane-associated protein

• Antibody selection and binding:

  • Test multiple antibodies (monoclonal antibodies like Santa Cruz's 1G8 clone show good IP performance)

  • Pre-clear lysate with protein A/G beads

  • Optimal antibody-to-lysate ratio typically 2-5 μg antibody per 500 μg protein

  • Allow extended binding time (overnight at 4°C)

• Washing considerations:

  • Use progressively stringent wash buffers

  • Perform 4-5 washes to minimize background

  • Maintain detergent concentration to prevent protein precipitation

• Elution strategies:

  • Gentle elution with excess peptide antigen (if available)

  • Standard SDS elution at 70°C rather than 95°C to minimize aggregation

  • For co-IP studies, consider crosslinking antibody to beads

• Verification methods:

  • Western blot with a different antibody than used for IP

  • Mass spectrometry for unbiased identification

  • Include IgG control and input samples for comparison

How can researchers optimize Western blot conditions for detecting low-abundance CH25H/ch25hl2?

For detecting low-abundance CH25H protein:

• Sample preparation optimization:

  • Enrich membrane fractions where CH25H localizes

  • Consider immunoprecipitation before Western blot

  • Use protease and phosphatase inhibitors during extraction

  • Avoid repeated freeze-thaw cycles

• Protein loading and transfer:

  • Increase protein loading (50-100 μg per lane)

  • Use PVDF membranes instead of nitrocellulose for better retention

  • Optimize transfer conditions: lower voltage for longer time

  • Consider semi-dry transfer for more efficient transfer of membrane proteins

• Blocking and antibody incubation:

  • Test alternative blocking agents (5% BSA often works better than milk for phospho-epitopes)

  • Extended primary antibody incubation (overnight at 4°C)

  • Optimize antibody dilution (typically 1:500 to 1:1000 for CH25H antibodies)

  • Consider signal enhancers or tyramide signal amplification

• Detection system selection:

  • Use high-sensitivity ECL substrates

  • Consider fluorescent secondary antibodies with longer exposure

  • Use cooled CCD camera systems for better detection

• Positive controls:

  • Include overexpression lysate as positive control

  • Use interferon-treated cells (which upregulate CH25H)

  • Consider tissues known to express high levels (liver, macrophages)

What are the optimal approaches for developing custom monoclonal antibodies against CH25H/ch25hl2?

For developing custom monoclonal antibodies against CH25H, researchers should consider:

• Antigen design strategy:

  • Full-length protein is challenging due to multiple transmembrane domains

  • Designer peptides from extracellular/cytoplasmic domains are preferable

  • Recombinant fragments of hydrophilic regions

  • Consider species conservation for cross-reactivity if desired

• Immunization protocols:

  • Use multiple host species (mouse, rabbit, hamster) for diverse repertoires

  • Implement extended immunization schedules (10-12 weeks)

  • Analyze serum titers to determine optimal fusion timing

  • Working with hybridoma centers can provide expertise in this area

• Screening method selection:

  • Primary screen: ELISA against immunizing antigen

  • Secondary screen: Western blot against native protein

  • Tertiary screen: Application-specific testing (IHC, IF, etc.)

• Cloning and validation:

  • Subclone positive hybridomas at least twice for stability

  • Isotype determination for appropriate purification strategy

  • Specificity testing against related family members

  • Knockout/knockdown validation

• Production and maintenance:

  • Optimal culture conditions determination

  • Cryopreservation at multiple stages

  • Ascites production if large quantities needed

  • Consider recombinant antibody production for consistency

The hybridoma approach has been successfully used for many targets and can be accessed through university core facilities, like the Washington University Hybridoma Center which has over 35 years of experience generating custom monoclonal antibodies .

What strategies can improve CH25H antibody performance in multiplex immunofluorescence studies?

For successful multiplex immunofluorescence with CH25H antibodies:

• Antibody panel design:

  • Select antibodies from different host species

  • Check for cross-reactivity between secondary antibodies

  • Use directly conjugated primary antibodies when possible

  • Plan the sequence of antibody application based on epitope sensitivity

• Sample preparation optimization:

  • Test multiple fixation methods (4% PFA, methanol, acetone)

  • Optimize antigen retrieval conditions (heat-induced vs. enzymatic)

  • Consider tissue clearing techniques for thick sections

  • Use tyramide signal amplification for weak signals

• Signal separation strategies:

  • Sequential staining with careful stripping between rounds

  • Spectral unmixing for overlapping fluorophores

  • Multi-round imaging with antibody stripping/quenching

  • Consider advanced approaches like CODEX or Imaging Mass Cytometry

• Controls and validation:

  • Single-color controls for spectral overlap assessment

  • Fluorescence-minus-one (FMO) controls

  • Absorption controls to verify antibody specificity

  • Include positive and negative tissue controls

• Data analysis approaches:

  • Use automated image analysis software

  • Implement machine learning for cell classification

  • Quantify co-localization coefficients

  • Perform spatial relationship analyses

How does CH25H function in viral immunity compare across model organisms?

CH25H functions show both conservation and divergence across species:

Research comparing the functions of CH25H across species has revealed:

• The enzymatic activity producing 25-hydroxycholesterol is conserved, though substrate specificity may vary.

• In zebrafish, the gene underwent duplication, resulting in multiple orthologs (ch25hl1.1, ch25hl1.2, ch25hl2) which may have undergone subfunctionalization .

• The antiviral properties appear conserved, though the specific mechanisms and virus susceptibility profiles differ between species.

• Developmental roles may be more prominent in lower vertebrates compared to mammals.

When studying these orthologs, researchers should consider using antibodies specifically validated for their species of interest, as cross-reactivity between human and zebrafish antibodies is often limited .

What are the emerging techniques for studying CH25H-mediated oxysterol production in live cells?

Recent methodological advances for studying CH25H activity include:

• Biosensor development:

  • FRET-based sensors for oxysterol binding

  • Engineered transcriptional reporters responding to oxysterols

  • Fluorescent analogues of cholesterol to track metabolism

• Live-cell imaging approaches:

  • Integration of nanobody technology for real-time protein tracking

  • Correlative light and electron microscopy to precisely localize CH25H

  • Super-resolution techniques to visualize membrane microdomains

• Metabolic labeling strategies:

  • Isotope-labeled cholesterol precursors

  • Click chemistry for tracking newly synthesized sterols

  • Mass spectrometry imaging for spatial metabolite detection

• Genome engineering applications:

  • CRISPR-mediated tagging of endogenous CH25H

  • Optogenetic control of CH25H expression or localization

  • Rapid protein degradation systems to study acute effects

These techniques are enabling researchers to move beyond static measurements of CH25H function to understand the dynamic regulation of oxysterol production in response to various stimuli, particularly during viral infection.

How can researchers address nonspecific binding issues with CH25H antibodies?

Nonspecific binding with CH25H antibodies can be addressed through these approaches:

• Blocking optimization:

  • Test different blocking agents (BSA, casein, commercial blockers)

  • Increase blocking time and concentration

  • Include mild detergents in blocking buffer (0.05-0.1% Tween-20)

• Antibody dilution and incubation:

  • Increase antibody dilution (start with manufacturer's recommendation, then titrate)

  • Reduce incubation temperature (4°C rather than room temperature)

  • Add non-ionic detergents to antibody diluent

• Washing protocol enhancement:

  • Increase number of washes (minimum 3-5 washes)

  • Use higher detergent concentration in wash buffers

  • Extend wash duration for each step

• Sample preparation considerations:

  • Ensure complete lysis and denaturation for Western blot

  • Optimize fixation for immunohistochemistry

  • Pre-absorb antibody with non-specific proteins

• Controls and validation:

  • Include peptide competition controls

  • Use knockout/knockdown samples as negative controls

  • Process identically with isotype control antibodies

Researchers should note that CH25H is a membrane-associated protein, which can sometimes lead to higher background when using certain extraction methods or fixation protocols.

What strategies can overcome challenges in detecting different CH25H isoforms?

Detecting specific CH25H isoforms requires careful experimental design:

• Isoform-specific antibody selection:

  • Use antibodies raised against unique regions of each isoform

  • Consider custom antibody development for poorly characterized isoforms

  • Verify specificity using overexpression of individual isoforms

• Resolution enhancement:

  • Use gradient gels for better separation of similar molecular weight isoforms

  • Consider Phos-tag gels for phosphorylated isoforms

  • Optimize gel percentage and running conditions

• Molecular approaches:

  • RT-PCR with isoform-specific primers

  • RNA-seq analysis for transcript-level quantification

  • Expression of tagged isoforms for validation

• Enrichment strategies:

  • Subcellular fractionation based on differential localization

  • Immunoprecipitation with isoform-specific antibodies

  • Size-exclusion chromatography for complexes

• Post-translational modification analysis:

  • Phosphatase treatment to identify phosphorylated forms

  • Deglycosylation enzymes for glycosylated variants

  • Mass spectrometry for comprehensive PTM mapping

For zebrafish studies, it's particularly important to distinguish between the multiple orthologs (ch25hl1.1, ch25hl1.2, ch25hl2) which may have divergent functions and expression patterns .