CERK Antibody

What is CERK and what are its primary functions in cellular processes?

CERK (Ceramide Kinase) is a lipid kinase that specifically catalyzes the phosphorylation of ceramide to produce ceramide-1-phosphate (C1P). This enzyme plays vital roles in numerous biological functions and is implicated in several pathological conditions, including:

• Aberrant wound healing processes

• Obesity-diabetes pathophysiology

• Mesangio-proliferative kidney diseases

• Cancer progression and metastasis

• Neuroblastoma development

CERK exists in two isoforms with molecular weights of 60 kDa and 38 kDa, with the 38 kDa form most commonly observed in experimental contexts . The enzyme's activity influences cellular processes ranging from inflammatory responses to cell survival and proliferation.

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

Based on current validation data, CERK antibodies have been successfully employed in the following applications:

Researchers should note that optimal dilutions may vary based on specific experimental conditions and antibody lots. It is recommended that each reagent should be titrated in individual testing systems to obtain optimal results .

What tissue and species reactivity has been confirmed for commercial CERK antibodies?

Current commercial CERK antibodies demonstrate confirmed reactivity with:

Experimental validation has shown clear Western blot bands at the expected molecular weight (38 kDa) in mouse heart tissue lysates . Additionally, immunohistochemical analysis has confirmed specific staining patterns in mouse heart tissue sections using heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) .

What are the optimal antigen retrieval methods for CERK immunohistochemistry?

For optimal CERK detection in immunohistochemistry applications, the following antigen retrieval approaches have been validated:

• Primary recommendation: Heat-mediated antigen retrieval with TE buffer pH 9.0

• Alternative approach: Heat-mediated antigen retrieval with citrate buffer pH 6.0

The immunohistochemical analysis of paraffin-embedded mouse heart tissue slides using CERK antibody (25731-1-AP) at a dilution of 1:200 with heat-mediated antigen retrieval (Tris-EDTA buffer, pH 9.0) has demonstrated clear and specific staining patterns under both 10x and 40x magnification .

What are the proper storage conditions for maintaining CERK antibody stability?

To maintain optimal activity and stability of CERK antibodies, the following storage conditions are recommended:

• Store at -20°C

• Stable for one year after shipment when properly stored

• Aliquoting is unnecessary for -20°C storage for most formulations

• Some formulations contain 0.02% sodium azide and 50% glycerol pH 7.3

• Alternative formulations include Rabbit IgG in pH 7.4 PBS, 0.05% NaN3, 40% Glycerol

For antibodies in liquid form, avoid repeated freeze-thaw cycles, which can lead to protein denaturation and loss of activity.

How can CERK activity be accurately determined in experimental cell culture systems?

For precise measurement of CERK enzymatic activity in cell culture systems, researchers have established a fluorescence-based assay utilizing NBD-C6-ceramide as a substrate. The protocol involves:

• Preparation of cell lysates (50-100 μg protein)

• Mixing with reaction buffer containing:

  • 20 mM Hepes (pH 7.4)

  • 10 mM KCl

  • 15 mM MgCl₂

  • 15 mM CaCl₂

  • 10% glycerol

  • 1 mM DTT

  • 1 mM ATP

  • 10 μM NBD-C6-ceramide (N-hexanoyl-D-erythro-sphingosine)

• Incubation for 30 minutes in darkness

• Reaction termination with chloroform:methanol (2:1)

• Centrifugation at 280,800 g for 30 seconds

• Transfer of 100 μl of the upper aqueous phase to 96-well plates

• Addition of 100 μl dimethylformamide (DMF)

• Fluorescence measurement using 495 nm excitation and 520 nm emission filters

This assay can be used to compare CERK activity between control and experimental conditions, such as siRNA-mediated CERK knockdown studies in differentiated 3T3-L1 adipocytes .

What is the role of CERK in inflammatory responses and how can researchers investigate these pathways?

CERK plays a significant role in inflammatory responses, particularly in TNF-α-induced immune responses in monocytes. Research has demonstrated that:

• CERK is involved in ceramide metabolism that affects inflammatory signaling

• CERK phosphorylates ceramide to produce C1P, which participates in inflammatory processes

• Disruption of CERK function can modulate TNF-α-induced inflammatory responses

• CERK activity influences the expression of inflammatory cytokines including IL-1β and MCP-1

To investigate these pathways, researchers can implement:

• CERK knockdown experiments using siRNA in relevant cell models

• Western blotting with CERK antibodies to confirm protein expression levels

• Quantitative analysis of downstream inflammatory mediators

• Pharmacological inhibition of CERK activity

• Assessment of C1P levels as a marker of CERK function

• Analysis of inflammatory response genes after TNF-α stimulation in the presence/absence of CERK activity

The combination of these approaches provides a comprehensive understanding of CERK's role in inflammatory signaling networks.

How does CERK function relate to adipogenesis and metabolic disorders?

Research indicates that CERK plays a significant role in adipocyte differentiation and metabolic regulation:

• CERK is implicated in the pathophysiology of obesity-diabetes

• Studies in 3T3-L1 cell models demonstrate CERK involvement in adipogenesis

• siRNA-mediated knockdown of CERK affects adipocyte differentiation processes

• CERK activity can be measured in differentiating preadipocytes

To investigate CERK's role in adipogenesis, researchers can use the following methodological approach:

• Seed 3T3-L1 cells at 1.2 × 10⁵ cells/well in 6-well plates

• Differentiate cells with standard adipogenic cocktail in the presence or absence of CERK siRNA

• Harvest cells at different timepoints during differentiation

• Analyze CERK protein expression by Western blotting using 12% SDS-PAGE gels

• Measure CERK activity using the NBD-C6-ceramide assay

• Assess adipogenic marker proteins and lipid accumulation

This approach allows for correlation between CERK expression/activity and adipocyte differentiation status, providing insights into potential therapeutic targets for metabolic disorders.

What technical considerations are important when detecting different CERK isoforms?

CERK is known to exist in two isoforms with molecular weights of 60 kDa and 38 kDa . When designing experiments to detect and distinguish these isoforms, researchers should consider:

• Gel percentage selection:

  • Use 10-12% SDS-PAGE gels for optimal separation of both isoforms

  • Lower percentage gels (7-8%) may provide better resolution of the 60 kDa isoform

• Sample preparation:

  • Different tissue types may express isoforms at varying ratios

  • Mouse heart and liver tissues reliably express detectable CERK levels

  • Cell lysis buffers should contain appropriate protease inhibitors

• Western blot optimization:

  • Longer separation times may improve resolution between isoforms

  • Transfer conditions may need adjustment for efficient transfer of larger isoforms

  • The 38 kDa isoform is most commonly observed in experimental contexts

• Antibody selection:

  • Confirm whether the antibody recognizes epitopes present in both isoforms

  • Check antibody documentation for validation with specific isoforms

  • Consider using isoform-specific antibodies if available

Understanding these technical considerations ensures accurate detection and characterization of CERK isoforms in experimental systems.

What is the optimal Western blot protocol for CERK detection?

For optimal CERK detection using Western blotting, the following protocol has been validated:

• Sample preparation:

  • Harvest cells or tissue and lyse in ice-cold homogenization buffer

  • Load 20-40 μg of protein per well

• Gel electrophoresis:

  • Use 12% SDS-PAGE separating gels for optimal resolution

  • Run at constant voltage until sufficient separation is achieved

• Transfer:

  • Transfer proteins to nitrocellulose membranes using standard protocols

  • Verify transfer efficiency with Ponceau S staining

• Blocking:

  • Block membranes with 5% skim milk in TBS containing 0.1% Tween 20 for 1 hour

• Primary antibody incubation:

  • Dilute CERK antibody 1:500-1:1000 in TBS-0.1% Tween 20

  • Incubate overnight at 4°C

• Washing:

  • Wash three times with TBS-0.1% Tween 20

• Secondary antibody incubation:

  • Incubate with horseradish peroxidase-conjugated secondary antibody at 1:4000 dilution for 1 hour

• Detection:

  • Visualize bands using enhanced chemiluminescence

  • Analyze exposed films with appropriate software (e.g., ImageJ)

This protocol has successfully detected CERK in mouse heart tissue lysates, showing clear bands at the expected molecular weight of 38 kDa .

How can siRNA approaches be combined with CERK antibody detection to study protein function?

The combination of siRNA-mediated knockdown with antibody detection provides a powerful approach to study CERK function:

• siRNA transfection in cell models:

  • Seed cells at appropriate density (e.g., 1.2 × 10⁵ cells/well for 3T3-L1)

  • Transfect with CERK-specific siRNA following manufacturer protocols

  • Include appropriate negative controls (scrambled siRNA)

• Verification of knockdown:

  • Harvest cells 48-72 hours post-transfection

  • Perform Western blotting using CERK antibody (1:500-1:1000 dilution)

  • Quantify reduction in protein levels relative to control conditions

• Functional assays:

  • After confirming knockdown, proceed with functional assays

  • For adipogenesis studies, differentiate 3T3-L1 cells using standard protocols

  • For inflammatory response studies, stimulate with TNF-α or other appropriate agents

  • Monitor downstream effects using appropriate readouts

• CERK activity assessment:

  • Perform NBD-C6-ceramide assay to confirm reduction in enzymatic activity

  • Correlate activity reduction with protein level decrease

This integrated approach allows researchers to establish direct relationships between CERK expression levels and specific cellular functions .

What controls should be included when working with CERK antibodies?

To ensure experimental rigor and data reliability when working with CERK antibodies, the following controls should be incorporated:

• Positive tissue controls:

  • Mouse heart tissue (validated for both WB and IHC)

  • Mouse liver tissue (validated for WB)

  • Human breast cancer tissue (validated for IHC)

• Negative controls:

  • Secondary antibody only (omitting primary antibody)

  • Isotype control (irrelevant primary antibody of same host/isotype)

  • CERK knockout or knockdown samples when available

• Loading controls for Western blot:

  • Housekeeping proteins (β-actin, GAPDH, α-tubulin)

  • Total protein staining methods (Ponceau S, REVERT)

• Antibody validation controls:

  • Peptide competition assay to confirm specificity

  • Multiple antibodies targeting different epitopes

  • Recombinant CERK protein as a size reference

• Technical controls:

  • Concentration gradients to establish optimal antibody dilutions

  • Comparison of different antigen retrieval methods for IHC

Implementing these controls ensures that experimental results with CERK antibodies are reliable, reproducible, and correctly interpreted.

Why might CERK antibodies show bands at unexpected molecular weights?

Researchers may observe CERK bands at molecular weights that differ from the expected 38 kDa and 60 kDa. Several factors may contribute to this phenomenon:

• Post-translational modifications:

  • Phosphorylation can alter protein migration

  • Glycosylation may increase apparent molecular weight

  • Other modifications may affect electrophoretic mobility

• Proteolytic processing:

  • Sample preparation conditions may cause protein degradation

  • Physiological proteolytic processing in certain tissues

  • Incomplete protease inhibition during extraction

• Alternative splicing:

  • CERK has known isoforms (60 kDa and 38 kDa)

  • Additional splice variants may exist in specific tissues

• Technical factors:

  • Buffer composition affecting protein migration

  • Gel percentage affecting resolution

  • Non-specific binding of antibody to related proteins

• Sample-specific factors:

  • Species differences in CERK molecular weight

  • Tissue-specific expression of particular isoforms

  • Disease state affecting protein processing or modification

When unexpected bands are observed, researchers should perform validation experiments, including peptide competition assays, alternative antibodies, or mass spectrometry-based protein identification to confirm band identity.

How can inconsistent IHC staining patterns with CERK antibodies be addressed?

Inconsistent immunohistochemical staining with CERK antibodies may result from various factors. Troubleshooting approaches include:

• Optimization of antigen retrieval:

  • Compare heat-mediated retrieval with TE buffer pH 9.0 (recommended) versus citrate buffer pH 6.0 (alternative)

  • Adjust retrieval time and temperature

  • Test pressure cooker versus microwave methods

• Antibody dilution optimization:

  • Test serial dilutions within the recommended range (1:50-1:500)

  • Perform titration experiments to identify optimal concentration

• Fixation considerations:

  • Overfixation may mask epitopes

  • Evaluate different fixation protocols

  • Fresh frozen versus FFPE tissue comparison

• Signal amplification:

  • Implement polymer-based detection systems

  • Consider tyramide signal amplification for low-abundance targets

  • Biotin-free detection systems may reduce background

• Counterstaining optimization:

  • Adjust hematoxylin intensity

  • Consider alternative counterstains for specific applications

The immunohistochemical analysis of paraffin-embedded mouse heart tissue using CERK antibody (25731-1-AP) at a dilution of 1:200 with heat-mediated antigen retrieval in Tris-EDTA buffer (pH 9.0) has yielded consistent and specific staining patterns under both low (10x) and high (40x) magnification .

What approaches can be used to quantify CERK expression levels across experimental conditions?

Accurate quantification of CERK expression is essential for comparative studies. The following approaches are recommended:

• Western blot quantification:

  • Use digital imaging systems rather than film for wider dynamic range

  • Include concentration standards when possible

  • Normalize to appropriate loading controls

  • Use analysis software (ImageJ) with consistent background subtraction methods

• Immunohistochemistry quantification:

  • Digital image analysis of stained sections

  • H-score or Allred scoring systems for semi-quantitative assessment

  • Automated tissue analysis platforms with appropriate controls

  • Cell-type specific quantification in heterogeneous tissues

• ELISA-based quantification:

  • Commercial ELISA kits for CERK protein quantification

  • Sandwich ELISA development using validated antibody pairs

  • Standard curves with recombinant CERK protein

• Activity-based quantification:

  • NBD-C6-ceramide assay to measure enzymatic activity

  • Correlation of activity with protein expression levels

• mRNA-protein correlation:

  • RT-qPCR for CERK mRNA quantification

  • Correlation analysis between mRNA and protein levels

  • Consideration of post-transcriptional regulation

These quantification approaches, when properly controlled and validated, provide reliable comparative data on CERK expression across experimental conditions.

How can researchers distinguish between specific and non-specific binding of CERK antibodies?

Distinguishing specific from non-specific binding is crucial for accurate data interpretation. Recommended approaches include:

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Compare staining patterns with and without peptide competition

  • Specific signals should be markedly reduced after competition

• Multiple antibody verification:

  • Use multiple antibodies targeting different CERK epitopes

  • Concordant results increase confidence in specificity

  • Discordant results warrant further investigation

• Genetic approaches:

  • CERK knockdown/knockout validation

  • siRNA-treated samples should show reduced signal

  • CRISPR/Cas9-edited cell lines as definitive controls

• Signal pattern analysis:

  • Specific staining should match known subcellular localization

  • Consistent molecular weight bands in Western blot

  • Tissue distribution consistent with known CERK expression patterns

• Technical controls:

  • Secondary antibody-only controls

  • Isotype controls

  • Host pre-immune serum controls

These approaches collectively provide strong evidence for antibody specificity and increase confidence in experimental results.

How can CERK antibodies be used to investigate ceramide-related signaling pathways in disease models?

CERK antibodies are valuable tools for investigating ceramide metabolism in disease contexts:

• Cancer research applications:

  • CERK is implicated in cancer progression and metastasis

  • IHC analysis of tumor tissues (e.g., breast cancer) can reveal expression patterns

  • Correlation of CERK expression with clinical outcomes

  • Investigation of CERK as a potential therapeutic target

• Metabolic disease models:

  • CERK's role in obesity-diabetes pathophysiology

  • Analysis of adipose tissue samples from disease models

  • Correlation with metabolic parameters

  • Therapeutic intervention studies targeting ceramide metabolism

• Inflammatory conditions:

  • CERK regulates TNF-α-induced immune responses

  • Analysis of inflammatory cell populations

  • Correlation with inflammatory mediator expression

  • Temporal dynamics of CERK activation during inflammation

• Neurological disorders:

  • CERK involvement in neuroblastoma

  • Investigation in neurodegenerative disease models

  • Brain tissue analysis using optimized IHC protocols

  • Correlation with neurological function and pathology

• Multi-omics integration:

  • Combination with lipidomics analysis of ceramide species

  • Integration with transcriptomics data

  • Correlation with proteomics profiling of related pathways

These research applications highlight the versatility of CERK antibodies in advancing our understanding of ceramide metabolism in health and disease.

What are the latest technical advances in CERK antibody-based detection methods?

Emerging technologies are enhancing the utility of CERK antibodies in research:

• Multiplexed immunofluorescence:

  • Simultaneous detection of CERK with other pathway components

  • Spectral unmixing for increased multiplexing capacity

  • Single-cell analysis of ceramide metabolism in heterogeneous tissues

• Proximity ligation assays:

  • Detection of protein-protein interactions involving CERK

  • Enhanced sensitivity for low-abundance interactions

  • In situ visualization of molecular complexes

• Mass cytometry (CyTOF):

  • Metal-conjugated antibodies for high-parameter analysis

  • Single-cell resolution of ceramide pathway components

  • Integration with other cellular parameters

• Super-resolution microscopy:

  • Nanoscale localization of CERK in cellular compartments

  • Co-localization with lipid domains and organelles

  • Dynamic tracking of CERK trafficking

• Automated quantitative analysis:

  • AI-assisted image analysis for IHC quantification

  • High-throughput screening applications

  • Standardized scoring algorithms for clinical translation

These technological advances are expanding the research applications of CERK antibodies and enabling more sophisticated investigations of ceramide metabolism and signaling.