mkrn2 Antibody

What is MKRN2 and what are its key biological functions?

MKRN2 (also known as RNF62 or HSPC070) is an E3 ubiquitin ligase that catalyzes the covalent attachment of ubiquitin moieties onto substrate proteins. With a molecular mass of approximately 47-49 kDa, MKRN2 contains four zinc finger domains and one RING finger domain that is critical for its ubiquitin ligase activity .

Key biological functions include:

• Negative regulation of NF-κB-mediated inflammatory responses through polyubiquitination and degradation of p65

• Suppression of non-small-cell lung cancer (NSCLC) cell migration and invasion via downregulation of the PI3K/Akt pathway

• Contribution to male fertility, with knockout models demonstrating infertility in male mice

• Involvement in regulating STAT1 expression levels

How should I select the appropriate MKRN2 antibody for my experimental needs?

Selection should be based on multiple factors:

Consider:

• The species being studied (human MKRN2 antibodies may not cross-react with all rodent models)

• Subcellular localization requirements (MKRN2 has been observed in both nuclear and cytoplasmic compartments)

• Validation status (look for antibodies validated using knockout controls)

• The epitope location (N-terminal, C-terminal, or internal sequences can affect detection depending on potential protein modifications or cleavage)

What are the optimal conditions for using MKRN2 antibodies in Western blotting?

For optimal Western blot results:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors and phosphatase inhibitors

  • Include N-ethylmaleimide (NEM) to preserve ubiquitinated protein species

  • Load 20-30 μg of total protein per lane

• Separation conditions:

  • 10-12% SDS-PAGE gels are recommended

  • The observed molecular weight is approximately 47 kDa

• Transfer and detection:

  • PVDF membranes are preferable over nitrocellulose

  • Blocking with 5% non-fat milk for 1 hour at room temperature

  • Primary antibody incubation at dilutions of 1:2000-1:16000 in 5% BSA solution overnight at 4°C

  • Secondary antibody incubation at 1:5000 for 1 hour at room temperature

  • Enhanced chemiluminescence detection with standard exposure times of 1-5 minutes

• Controls:

  • Include positive control lysates (HeLa, A375, or K-562 cells have been validated)

  • Consider using MKRN2 knockout/knockdown samples as negative controls

How can I optimize immunohistochemistry protocols for MKRN2 detection in tissue samples?

For successful IHC detection of MKRN2:

• Fixation and processing:

  • 10% neutral buffered formalin fixation for 24-48 hours

  • Standard paraffin embedding and sectioning at 4-5 μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval is essential

  • Primary recommendation: TE buffer (pH 9.0) at 95-98°C for 15-20 minutes

  • Alternative: Citrate buffer (pH 6.0) if TE buffer yields high background

• Blocking and antibody incubation:

  • 3% H₂O₂ for 10 minutes to block endogenous peroxidase

  • Protein blocking with 5% normal serum for 1 hour

  • Primary antibody at 1:50-1:500 dilution (start with 1:100) overnight at 4°C

  • Detection with appropriate polymer/secondary system (such as EliVision Reagent)

  • Development with 3,3-diaminobenzidine tetrahydrochloride

• Evaluation:

  • MKRN2 is localized in both cytoplasm and nucleus

  • Scoring systems should account for both staining intensity and percentage of positive cells

What approaches should be used to verify MKRN2 antibody specificity?

Multiple validation strategies should be employed:

• Genetic validation:

  • MKRN2 knockout/knockdown models serve as gold standard negative controls

  • siRNA-mediated knockdown can confirm specificity in cell culture models

• Peptide competition:

  • Pre-incubation of antibody with immunizing peptide should abolish specific signal

• Orthogonal validation:

  • Multiple antibodies targeting different epitopes should show consistent patterns

  • Correlation of protein detection with mRNA expression data

• Cross-reactivity assessment:

  • Testing in species with known sequence homology

  • Evaluating potential cross-reactivity with related proteins (e.g., other MKRN family members)

• Application-specific controls:

  • For co-IP experiments, include IgG controls and reverse immunoprecipitation

  • For IF/ICC, include secondary-only controls and subcellular marker co-staining

How can MKRN2 antibodies be utilized to study its E3 ligase activity and substrate interactions?

To investigate MKRN2's E3 ligase activity:

• In vitro ubiquitination assays:

  • Purify MKRN2 protein by immunoprecipitation from cells transfected with MKRN2 expression plasmid

  • Combine with recombinant p65 (or other substrate), E1, E2, and biotinylated-ubiquitin in reaction buffer

  • Detect ubiquitinated substrate using Western blot with appropriate antibodies

• Substrate identification:

  • Perform immunoprecipitation with MKRN2 antibodies followed by mass spectrometry

  • Use MKRN2 antibodies in proximity ligation assays to detect in situ interactions

  • For known substrates like p65 or PI3Kp85α, co-immunoprecipitation experiments with tagged constructs can confirm interaction

• Domain-function analysis:

  • Compare wild-type MKRN2 with RING domain mutants (MKRN2-ΔRING) to assess E3 ligase function

  • The RING domain is essential for ubiquitin ligase activity but not for substrate recognition

• Cellular compartment fractionation:

  • Separate soluble and insoluble nuclear fractions to track substrate movement

  • MKRN2 can shuttle p65 from soluble to insoluble nuclear compartments prior to degradation

What methodological approaches are recommended for studying MKRN2's role in inflammatory regulation?

For investigating MKRN2's role in inflammatory regulation:

• NF-κB activity assessment:

  • NF-κB luciferase reporter assays with varying MKRN2 expression levels

  • Analysis of p65 nuclear translocation by immunofluorescence or subcellular fractionation

  • Measurement of NF-κB target gene expression by qRT-PCR

• Dendritic cell models:

  • Bone marrow-derived dendritic cells with MKRN2 knockdown show enhanced pro-inflammatory responses

  • Analyze nuclear p65 levels after LPS stimulation

  • Measure cytokine production (IL-6, IL-12, TNFα) in response to TLR ligands

• MKRN2-PDLIM2 cooperative mechanism:

  • Co-immunoprecipitation to confirm MKRN2-PDLIM2 interaction

  • Assess their synergistic effects on p65 polyubiquitination

  • Compare effects of individual versus combined knockdown

• Analysis of domain-specific functions:

  • Use deletion mutants (e.g., MKRN2-ΔRING) to separate p65 degradation from sequestration effects

  • The RING finger domain mediates ubiquitination, while other domains may be involved in protein-protein interactions

How can researchers effectively study MKRN2's involvement in cancer progression?

To investigate MKRN2's role in cancer:

• Expression analysis in tumor specimens:

  • IHC staining of MKRN2 in cancer tissues compared to adjacent normal tissues

  • Scoring based on staining intensity and percentage of positive cells

  • Correlation with clinicopathological parameters (differentiation, lymph node metastasis, TNM stage)

• Functional assays in cancer cell lines:

  • Modulate MKRN2 expression through overexpression or knockdown

  • Cell migration assays (wound healing, transwell)

  • Invasion assays (Matrigel-coated transwell)

  • Assessment of PI3K/Akt pathway activation (phosphorylation status of Akt)

• Mechanistic studies:

  • Immunoprecipitation to detect MKRN2's interaction with PI3Kp85α

  • In vitro ubiquitination assays to confirm ubiquitin-dependent degradation

  • Rescue experiments with pathway inhibitors or constitutively active constructs

• Prognostic correlation:

  • Kaplan-Meier survival analysis based on MKRN2 expression levels

  • Multivariate analysis to determine if MKRN2 is an independent prognostic factor

What are common challenges when using MKRN2 antibodies, and how can they be addressed?

Common challenges and solutions:

• High background in immunohistochemistry:

  • Optimize antibody dilution (start with higher dilutions and titrate)

  • Ensure adequate blocking (extend blocking time or increase blocker concentration)

  • Try alternative antigen retrieval methods (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Consider using more specific detection systems with lower cross-reactivity

• Multiple bands in Western blot:

  • Possible explanations include post-translational modifications, splice variants, degradation products

  • Use fresh samples with complete protease inhibitor cocktails

  • Include knockout or knockdown controls to identify specific bands

  • Consider using denaturing conditions that disrupt protein-protein interactions

• Weak signal in immunoprecipitation:

  • Increase starting material (1 mg protein recommended for IP)

  • Optimize antibody-to-sample ratio

  • Extend incubation time (overnight at 4°C)

  • Use more sensitive detection methods for Western blotting of IP samples

• Variability in MKRN2 subcellular localization:

  • MKRN2 can be found in both cytoplasm and nucleus, with cell-type specific patterns

  • Primary fibroblasts and HeLa cells show predominantly nuclear localization

  • Validate compartmentalization with subcellular fractionation and multiple microscopy methods

How should researchers interpret conflicting results regarding MKRN2 expression or function?

When facing conflicting results:

• Consider experimental context differences:

  • Cell/tissue type specificity (MKRN2 functions may differ between cell types)

  • Disease state (inflammatory conditions may alter MKRN2's activity or localization)

  • Species differences (mouse vs. human MKRN2 may have subtle functional distinctions)

• Antibody-related considerations:

  • Antibodies targeting different epitopes may yield different results

  • Batch-to-batch variation can occur even with the same catalog number

  • Compare results from multiple independent antibodies

• Technical approaches:

  • Validate protein expression with orthogonal methods (Western blot, IF, IHC, mass spectrometry)

  • Correlate protein detection with mRNA expression (qRT-PCR, RNA-seq)

  • Use genetic models (knockout, knockdown, overexpression) for functional validation

• Integrate multiple lines of evidence:

  • MKRN2 has been implicated in multiple pathways (NF-κB, PI3K/Akt, STAT1)

  • Seemingly contradictory results may reflect context-dependent roles in different pathways

  • Consider feedback loops and compensatory mechanisms in different experimental systems

What are the emerging research questions regarding MKRN2's role in disease pathogenesis?

Frontier research questions include:

• Cancer biology:

  • How does MKRN2's tumor-suppressive role in NSCLC reconcile with potential roles in other cancer types?

  • Does MKRN2 expression correlate with response to specific cancer therapies?

  • Can MKRN2 status serve as a predictive biomarker for immunotherapy response?

• Inflammatory disorders:

  • How does MKRN2 interact with other negative regulators of NF-κB signaling?

  • Is MKRN2 dysregulation involved in autoimmune or chronic inflammatory diseases?

  • Could targeting MKRN2 provide therapeutic benefit in inflammatory conditions?

• Reproductive biology:

  • What is the precise mechanism through which MKRN2 contributes to male fertility?

  • How does MKRN2 regulate STAT1, SIX4, and TNC expression in testicular tissue?

  • Is MKRN2 dysregulation associated with human male infertility disorders?

• Neurobiology:

  • Does MKRN2's reported function in negatively regulating neurogenesis via PI3K/Akt signaling have implications for neurodevelopmental disorders?

  • What is MKRN2's role in neuroinflammatory processes?

What methodological innovations might advance MKRN2 research?

Emerging methodologies with potential impact:

• Proximity-dependent approaches:

  • BioID or TurboID-MKRN2 fusion proteins to identify proximal interacting partners

  • Proximity ligation assays to visualize and quantify MKRN2-substrate interactions in situ

• Single-cell analysis:

  • Single-cell proteomics to characterize MKRN2 expression heterogeneity within tissues

  • Single-cell RNA-seq to correlate MKRN2 with target gene expression at individual cell resolution

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize MKRN2's precise subcellular localization

  • Live-cell imaging with fluorescently tagged MKRN2 to track dynamic responses to stimuli

• CRISPR-based screening:

  • CRISPR activation/interference screens to identify genes that modify MKRN2 phenotypes

  • CRISPR base editing to introduce specific mutations in MKRN2 RING domain or zinc fingers

• Structural biology:

  • Cryo-EM studies of MKRN2 in complex with substrate proteins

  • Small-molecule screening for compounds that modulate MKRN2's E3 ligase activity

What are the key considerations for researchers beginning work with MKRN2 antibodies?

For those initiating MKRN2 research:

• Antibody selection and validation:

  • Begin with well-validated antibodies cited in peer-reviewed publications

  • Always include appropriate positive and negative controls

  • Validate in your specific experimental system using genetic approaches when possible

• Experimental design:

  • Consider MKRN2's dual cytoplasmic and nuclear localization

  • Include proteasome inhibitors (e.g., MG132) when studying degradation mechanisms

  • Design experiments to distinguish between ubiquitination, sequestration, and degradation effects

• Pathway analysis:

  • Examine multiple pathways potentially affected by MKRN2 (NF-κB, PI3K/Akt, STAT1)

  • Use pathway-specific readouts beyond simple protein levels (luciferase reporters, target gene expression)

  • Consider context-dependent effects based on cell type and stimulus

• Interpretation guidance:

  • MKRN2 may have different functions depending on cell type and physiological context

  • Integrate protein-level data with transcriptomic and functional readouts

  • Consider compensatory mechanisms that may mask phenotypes in long-term studies