MKRN1 Antibody, Biotin conjugated

What is MKRN1 and what are its primary biological functions?

MKRN1 (Makorin Ring Finger Protein 1) is a conserved RNA-binding E3 ubiquitin ligase with multiple biological functions. It plays critical roles in several cellular processes:

• Functions as an E3 ubiquitin ligase that targets specific proteins for degradation, notably AMPKα subunits, affecting energy metabolism

• Promotes ribosome stalling at poly(A) sequences as part of ribosome-associated quality control (RQC) mechanisms

• Associates with ribosomes and is present in polysomal fractions, co-sedimenting with PABPC1

• Operates as a component of mRNPs (messenger ribonucleoprotein particles) in embryonic stem cells

• Predominantly associates with proteins involved in mRNA metabolism, including regulators of mRNA turnover, transport, and translation

• Affects gastric tumorigenesis by repressing cellular senescence and tumor-suppressive effects

MKRN1 contains several RNA-binding domains alongside its RING domain, which confers E3 ligase activity, enabling it to function at the intersection of RNA metabolism and protein degradation pathways.

What applications are biotin-conjugated MKRN1 antibodies suitable for?

Based on available product information, biotin-conjugated MKRN1 antibodies are specifically validated for:

• Immunohistochemistry (IHC)

• Immunohistochemistry with paraffin-embedded sections (IHC-p)

• Immunoprecipitation (IP)

While unconjugated MKRN1 antibodies have been validated for additional applications:

How should researchers validate the specificity of MKRN1 antibodies?

Rigorous validation is essential for confident interpretation of experimental results. Based on published methodologies:

• Perform immunoblot analysis comparing MKRN1 overexpression and knockdown cell lines

• Compare detection of FLAG-epitope-tagged recombinant MKRN1 protein with endogenous MKRN1

• Verify reduction of signal in lysates derived from stable MKRN1 knockdown cells

• Use MKRN1 siRNA (#5, #6, #7) or shRNA-expressing lentiviral particles to generate knockdown controls

• Include protein expression analysis alongside mRNA level assessment to differentiate between transcriptional and post-translational regulation

As reported: "The specificity of the MKRN1 antibody used to test the dynamics of MKRN1 expression was confirmed by immunoblot analysis of MKRN1 overexpression and knockdown ESC clones" .

What species reactivity has been confirmed for MKRN1 antibodies?

MKRN1 antibodies show varying cross-reactivity profiles:

This diversity enables research across multiple model systems, with broader cross-reactivity generally observed with polyclonal antibodies targeting conserved regions.

What experimental controls are essential when using biotin-conjugated MKRN1 antibodies?

To ensure reliable results, include the following controls:

• Positive controls: Cell lines with confirmed MKRN1 expression, such as OCT4+ undifferentiated embryonic stem cells, which show high MKRN1 expression

• Negative controls: MKRN1 knockdown samples using verified siRNA (#5: 5′-CAGGCGAAGCTGAGTCAAGAA-3′, #6: 5′-CGGGATCCTCTCCAACTGCAA-3′, #7: 5′-caggcgaagctgagtcaag-3′)

• Endogenous biotin blocking: Critical for biotin-conjugated antibodies to prevent non-specific binding to endogenous biotin-containing proteins

• Isotype controls: Match the primary antibody isotype (e.g., IgG2b for monoclonal antibodies)

• Secondary-only controls: To assess background from detection reagents

• Absorption controls: Pre-incubation of antibody with immunizing peptide to confirm specificity

High-content imaging analysis and flow cytometry quantification of MKRN1 expression provide additional validation approaches, as demonstrated in published research .

What methodological considerations are important for immunohistochemistry with biotin-conjugated MKRN1 antibodies?

For optimal IHC and IHC-p results with biotin-conjugated MKRN1 antibodies:

• Antigen retrieval: Heat-induced epitope retrieval is likely necessary for paraffin sections

• Endogenous biotin blocking: Use commercial biotin/avidin blocking kits, particularly crucial for biotin-rich tissues (liver, kidney)

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

• Detection system: Streptavidin-HRP followed by DAB or AEC for chromogenic detection; streptavidin-fluorophore conjugates for fluorescent detection

• Counterstaining: Hematoxylin for chromogenic detection; DAPI for fluorescent nuclear counterstaining

• Mounting: Use non-fluorescent mounting media for fluorescent detection to prevent photobleaching

The biotin-conjugated MKRN1 antibody (NB100-55304B) is specifically validated for IHC and IHC-p applications .

How can MKRN1 antibodies be used to investigate protein-protein interactions?

To elucidate MKRN1's interactome:

• Co-immunoprecipitation (Co-IP): Use MKRN1 antibodies to pull down protein complexes, followed by immunoblotting for suspected interaction partners

• Proximity ligation assay (PLA): Detect in situ protein-protein interactions with spatial resolution

• Affinity purification-mass spectrometry (AP-MS): Combined with MKRN1 antibodies for comprehensive interactome mapping

• Yeast two-hybrid screening: Complementary approach to identify direct binding partners

Published research identified 48 proteins consistently enriched in FLAG:MKRN1 purifications through LC-MS/MS analysis (FDR < 0.05), with 35 of these being established RNA-binding proteins and/or components of ribonucleoprotein complexes .

What methodological approaches can detect MKRN1 expression at the single-cell level?

For high-resolution analysis of MKRN1 expression:

• Flow cytometry: Quantify mean fluorescence intensity of MKRN1 at the single-cell level, as demonstrated in studies comparing OCT4+ and OCT4- cell populations

• High-content imaging: Correlate MKRN1 expression with other markers at the single-cell level

• Immunofluorescence microscopy: Visualize subcellular localization and co-localization with other proteins

• Mass cytometry (CyTOF): Simultaneous detection of multiple proteins including MKRN1 without spectral overlap concerns

Research has shown that "MKRN1 was most abundant in the OCT4+ cells of the ESC colony, and comparatively diminished in the OCT4− cells proximal to the colonies" , demonstrating differential expression patterns detectable at single-cell resolution.

How can biotin-conjugated MKRN1 antibodies help investigate its role in ribosome-associated quality control?

MKRN1 has been identified as a factor in ribosome-associated quality control (RQC), specifically promoting ribosome stalling at poly(A) sequences . To investigate this function:

• Polysome profiling: Fractionate ribosomes on sucrose gradients and detect MKRN1 distribution using biotin-conjugated antibodies

• Ribosome stalling assays: Employ dual fluorescence reporters to monitor ribosome stalling at poly(A) sequences with and without MKRN1

• Ribosome footprinting: Analyze ribosome positioning at poly(A) sequences in MKRN1-depleted versus control cells

• RNA-protein crosslinking: Identify direct MKRN1 binding sites in relation to ribosome positioning

MKRN1 "co-sedimented with PABPC1, indicating that together with PABPC1, MKRN1 is associated with translating ribosomes" , making these approaches suitable for examining its regulatory role in translation.

What strategies can elucidate MKRN1's E3 ubiquitin ligase activity using antibodies?

To investigate MKRN1's E3 ligase function:

• In vitro ubiquitination assays: Reconstitute ubiquitination reactions with purified components

• Ubiquitination target identification: Immunoprecipitate MKRN1 targets (e.g., AMPKα) and probe for ubiquitin modifications

• Functional comparison: Compare wild-type MKRN1 with E3 ligase-defective mutants (e.g., MKRN1 H307E)

• Proteasome inhibition: Treat cells with MG132 to accumulate ubiquitinated substrates before immunoprecipitation

• Mass spectrometry: Identify ubiquitination sites on MKRN1 substrates

Research has demonstrated that "AMPKα stabilised by MKRN1 depletion was degraded upon overexpression of MKRN1 resistant to MKRN1-targeting siRNA but was not affected by the expression of MKRN1 H307E, an E3 ligase-defective MKRN1 mutant" .

How can MKRN1 antibodies facilitate research on energy metabolism regulation?

MKRN1 regulates energy metabolism through control of AMPK stability. To investigate this function:

• Tissue-specific expression analysis: Examine MKRN1 distribution in metabolically active tissues

• Metabolic challenge experiments: Monitor MKRN1 expression changes in response to fasting, high-fat diet, or exercise

• Co-immunoprecipitation: Confirm MKRN1-AMPK interactions under various metabolic conditions

• Phospho-specific immunoblotting: Analyze AMPK phosphorylation status (T172) in relation to MKRN1 levels

Studies have shown that "MKRN1 depletion promotes glucose consumption and suppresses lipid accumulation due to AMPK stabilisation and activation. Accordingly, MKRN1-null MEFs increased the levels of activated AMPKα (phosphorylated at T172), which suppressed the activity of its downstream target, acetyl coenzyme A ACC, via phosphorylation" .

What techniques can explore MKRN1's RNA-binding properties?

To investigate MKRN1's interactions with RNA:

• CLIP-seq: Cross-linking immunoprecipitation followed by sequencing to identify RNA binding sites at nucleotide resolution

• RIP-seq: RNA immunoprecipitation to isolate and sequence MKRN1-bound RNAs

• PAR-CLIP: Photoreactive nucleoside-enhanced crosslinking for enhanced RNA-protein interaction mapping

• RNA pull-down assays: Using biotinylated RNA baits and MKRN1 detection by immunoblotting

Research has revealed that "MKRN1 binding sites were massively enriched in AAAA tetramers (A, adenosine) within 5–50 nucleotides (nt) downstream of the binding sites" and "30% (1848 out of 6165) of MKRN1 binding sites resided immediately upstream of an A-rich stretch" .

What are common sources of false positives/negatives when using biotin-conjugated MKRN1 antibodies?

Testing antibody specificity as done in published research: "bands corresponding to FLAG-epitope-tagged recombinant MKRN1 protein (FLAG:MKRN1) were uniquely detected from stable MKRN1 overexpression ESC clones, while bands representing endogenous MKRN1 were visibly reduced in lysates derived from stable MKRN1 knockdown ESC clones" .

How should researchers interpret conflicting results from different MKRN1 antibodies?

When faced with discrepancies between different MKRN1 antibodies:

• Compare epitope regions: Different antibodies target distinct regions (N-terminal, C-terminal, or internal domains)

• Consider isoform specificity: Some antibodies may recognize specific MKRN1 isoforms but not others

• Evaluate post-translational modifications: Modifications may mask epitopes in context-dependent manner

• Assess antibody validation: Review validation data (knockdown/overexpression controls)

• Cross-validate: Use orthogonal techniques (mass spectrometry, RNA interference) to confirm findings

The variety of available MKRN1 antibodies with different epitope specificities necessitates careful selection and validation for each experimental context.

What factors influence the detection of MKRN1 in different subcellular compartments?

MKRN1 localization can vary depending on:

• Cellular context: MKRN1 is preferentially expressed in undifferentiated (OCT4+) versus differentiated (OCT4-) cells

• Stress conditions: MKRN1 is recruited to stress granules upon exposure to environmental stress

• Protein interactions: Association with PABPC1 and other RNA-binding proteins may influence localization

• Cell cycle stage: Expression and localization may vary throughout the cell cycle

• Fixation method: Different fixatives may preserve or mask distinct epitopes

Research shows that "MKRN1 is preferentially expressed in the undifferentiated fraction of ESCs" and "MKRN1 protein expression was significantly more abundant in the OCT4+ cells than the OCT4− subpopulation" .

How can quantitative MKRN1 expression data be properly analyzed and interpreted?

For robust quantitative analysis:

• Normalization: Normalize MKRN1 signal to appropriate housekeeping controls

• Single-cell quantification: Use flow cytometry or high-content imaging for population heterogeneity analysis

• Statistical approaches: Apply appropriate statistical tests (t-test for two conditions, ANOVA for multiple conditions)

• Correlation analysis: Examine relationships between MKRN1 expression and other markers (e.g., OCT4)

• Dynamic range assessment: Include positive and negative controls to establish the assay's detection limits

Published methodologies include "the mean fluorescent intensity of MKRN1 in the OCT4+ and OCT4− fraction of ESC populations cultured for 72 h in self-renewal (+LIF) or differentiation conditions (−LIF or +RA) was quantified at the single-cell level using flow cytometry" .

How are MKRN1 antibodies contributing to understanding its role in disease pathogenesis?

MKRN1 has been implicated in several pathological processes:

• Cancer biology: MKRN1 affects gastric tumorigenesis by repressing cellular senescence and tumor-suppressive effects

• Metabolic disorders: Through its regulation of AMPK, MKRN1 influences energy metabolism pathways

• Translational quality control: As a factor in ribosome-associated quality control, MKRN1 may impact diseases related to protein misfolding

Studies investigating MKRN1's role in gastric cancer found that "MKRN1 could affect gastric tumorigenesis by repressing cellular senescence and tumor-suppressive effects" , highlighting its potential significance in cancer research.

What emerging techniques might enhance MKRN1 antibody applications in research?

Advanced methodologies that could expand MKRN1 research include:

• Super-resolution microscopy: Visualize MKRN1 localization at nanoscale resolution

• CRISPR-based tagging: Endogenous tagging of MKRN1 for live-cell imaging

• Mass spectrometry imaging: Map MKRN1 distribution in tissue sections

• Single-cell proteomics: Analyze MKRN1 expression and modification states at single-cell resolution

• Highly multiplexed imaging: Examine MKRN1 in relation to dozens of other proteins simultaneously

These approaches would complement existing methods used in MKRN1 research, such as iCLIP for mapping RNA-binding sites and AP-MS for interactome analysis .