MKLN1 Antibody, HRP conjugated

What is MKLN1 and why is it a target for antibody development?

MKLN1 (Muskelin 1, Intracellular Mediator Containing Kelch Motifs) is a cytoplasmic protein involved in cellular processes including intracellular transport and cytoskeletal organization. It contains distinctive kelch motifs that form β-propeller structures important for protein-protein interactions. MKLN1 has emerged as a significant research target due to its potential roles in cellular trafficking pathways and possible involvement in disease processes. Recent studies suggest MKLN1 functions as a CTLH Ubiquitin Ligase substrate, indicating its regulated degradation may be important in cellular homeostasis . Antibodies targeting MKLN1 provide valuable tools to study its expression, localization, and functional interactions in various biological contexts, particularly in research examining cellular trafficking mechanisms and protein degradation pathways.

What are the key specifications of commercially available MKLN1 antibodies with HRP conjugation?

The MKLN1 antibody conjugated to HRP (horseradish peroxidase) is typically a polyclonal antibody raised in rabbits against specific amino acid sequences of human MKLN1. A commonly referenced variant targets amino acids 488-614 of MKLN1 (catalog ABIN7160372) . This antibody demonstrates specific reactivity to human MKLN1 and has been validated for applications including ELISA. The antibody undergoes Protein G purification with purity exceeding 95% . The HRP conjugation provides direct enzymatic detection capability, eliminating the need for secondary antibodies in many applications. The immunogen used for production is a recombinant human Muskelin protein fragment encompassing amino acids 488-614 .

How does the epitope targeting (AA 488-614) of this antibody influence its applications?

The epitope targeting of amino acids 488-614 in MKLN1 has important implications for antibody applications. This region falls within the central portion of the MKLN1 protein, away from both the N-terminal and C-terminal regions which may have distinct functional domains. This positioning may provide several advantages: (1) The targeted region may be more accessible in the native protein conformation, enhancing detection sensitivity in applications like ELISA and Western blotting; (2) This region appears to be immunogenic and well-conserved in human MKLN1, contributing to antibody specificity; (3) The epitope avoids the kelch-repeat containing regions that might share homology with other kelch-domain proteins, potentially reducing cross-reactivity . Researchers should consider this epitope specificity when designing experiments, particularly when studying protein-protein interactions or conformational changes that might mask or alter this region of MKLN1.

What are the optimal protocols for using HRP-conjugated MKLN1 antibodies in Western blotting applications?

For Western blotting applications using HRP-conjugated MKLN1 antibodies, the following protocol optimizations are recommended:

Sample preparation:

• Extract proteins using RIPA buffer supplemented with protease inhibitors

• Load 20-40 μg of total protein per lane

• Include phosphatase inhibitors if studying phosphorylation states

Electrophoresis and transfer:

• Use 10% SDS-PAGE gels for optimal resolution of MKLN1 (~735 amino acids, ~80 kDa)

• Transfer to PVDF membranes at 100V for 90 minutes in cold transfer buffer containing 20% methanol

Antibody incubation:

• Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Dilute HRP-conjugated MKLN1 antibody 1:1000 to 1:2000 in blocking solution

• Incubate overnight at 4°C with gentle agitation

• Wash 4-5 times with TBST, 5 minutes each

Detection:

• Use enhanced chemiluminescence (ECL) substrate appropriate for HRP

• Initial exposure times of 30 seconds, 1 minute, and 5 minutes are recommended

• Expected band size for human MKLN1 is approximately 80 kDa

Controls:

• Include positive control lysates from cell lines known to express MKLN1 (HeLa cells are often suitable)

• Consider running a peptide competition assay to confirm specificity

• Include loading controls (β-actin, GAPDH) on the same blot

This protocol leverages the direct HRP conjugation to eliminate the secondary antibody step, reducing background and cross-reactivity issues while potentially enhancing sensitivity .

How can researchers optimize ELISA protocols using HRP-conjugated MKLN1 antibodies?

ELISA optimization with HRP-conjugated MKLN1 antibodies requires careful consideration of several parameters:

Direct ELISA protocol:

• Coat plates with target antigen (recombinant MKLN1 or sample) at 1-10 μg/ml in carbonate buffer (pH 9.6), overnight at 4°C

• Block with 3% BSA in PBS for 2 hours at room temperature

• Dilute HRP-conjugated MKLN1 antibody (starting range: 1:500 to 1:5000) in blocking buffer

• Incubate for 2 hours at room temperature

• Wash 5 times with PBST (PBS + 0.05% Tween-20)

• Add TMB substrate and monitor color development

• Stop reaction with 2N H₂SO₄ and read absorbance at 450 nm

Sandwich ELISA considerations:

• For detecting native MKLN1, use an unconjugated capture antibody targeting a different epitope (e.g., N-terminal region)

• Apply HRP-conjugated MKLN1 antibody (AA 488-614) as the detection antibody

• Establish standard curves using recombinant MKLN1 protein (10 pg/ml to 1000 ng/ml)

Optimization parameters:

• Antibody titration: Perform checkerboard titration to determine optimal antibody concentration

• Sample dilution: Test serial dilutions to ensure linearity in the assay range

• Incubation temperature: Compare room temperature vs. 37°C incubation for signal enhancement

• Substrate incubation time: Monitor kinetics of color development (5-30 minutes)

Validation controls:

• Include recombinant MKLN1 protein as positive control

• Test antibody specificity using related kelch-domain proteins

• Evaluate precision through intra- and inter-assay coefficient of variation (<15% is generally acceptable)

These optimization steps will help establish a reliable ELISA protocol with appropriate sensitivity and specificity for MKLN1 detection using HRP-conjugated antibodies .

What are effective strategies for immunofluorescence applications that complement HRP-based detection methods?

While HRP-conjugated MKLN1 antibodies are primarily used for enzymatic detection methods, researchers often need complementary immunofluorescence approaches for co-localization studies. Based on approaches used with other antibody systems like anti-MUC1 antibodies, the following strategies are recommended:

Complementary immunofluorescence protocol:

• Fix cells with 4% paraformaldehyde (10 minutes) followed by permeabilization with 0.1% Triton X-100 (5 minutes)

• Block with 3% BSA in PBS for 30 minutes at room temperature

• Use unconjugated MKLN1 antibody targeting the same epitope (AA 488-614) at 1:100-1:500 dilution

• Incubate overnight at 4°C in a humidified chamber

• Apply fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488 anti-rabbit)

• Counterstain nucleus with DAPI and mount with anti-fade mounting medium

Advanced co-localization strategies:

• For dual labeling, combine MKLN1 antibody with markers for subcellular compartments (e.g., GM130 for Golgi, LAMP1 for lysosomes)

• When studying protein internalization dynamics, adapt methods from studies of other proteins by conjugating the primary antibody directly with fluorescent dyes like DyLight 488

• For live-cell imaging, consider using cell-permeable fluorescent-tagged MKLN1 antibody fragments

• Implement confocal microscopy with Z-stack acquisition to precisely determine three-dimensional localization

Validation approaches:

• Compare staining patterns with multiple antibodies targeting different MKLN1 epitopes

• Include appropriate controls: primary antibody omission, peptide competition, and siRNA knockdown samples

• Quantify co-localization using appropriate coefficients (Pearson's, Mander's) and statistical analysis

This approach leverages techniques demonstrated effective with other antibody systems such as anti-MUC1, where researchers successfully tracked protein internalization and localization through conjugation with fluorescent dyes .

How can researchers troubleshoot non-specific binding or high background when using HRP-conjugated MKLN1 antibodies?

Non-specific binding and high background are common challenges when working with HRP-conjugated antibodies. Based on experience with similar antibody systems, the following troubleshooting approaches are recommended:

Common causes and solutions for high background:

Advanced troubleshooting for MKLN1 specificity:

• Compare results using different MKLN1 antibodies targeting distinct epitopes (e.g., N-terminal region)

• Implement knockout/knockdown validation by analyzing samples from MKLN1-depleted cells

• For critical applications, consider performing immunoprecipitation followed by mass spectrometry to confirm antibody specificity

• When working with tissue samples, include appropriate tissue-specific negative controls

These approaches systematically address the most common causes of non-specific binding and high background, helping researchers achieve cleaner results with HRP-conjugated MKLN1 antibodies across different applications.

What are the key considerations for quantitative analysis of MKLN1 expression levels using HRP-conjugated antibodies?

Quantitative analysis of MKLN1 expression requires rigorous methodology to ensure accuracy and reproducibility. Consider these key factors:

Western blot quantification:

• Establish linear dynamic range by loading protein concentration gradients (5-50 μg)

• Use digital image acquisition systems rather than film for better quantitative accuracy

• Implement housekeeping protein controls (β-actin, GAPDH) that are appropriate for your experimental conditions

• Analyze band intensity using software (ImageJ, Image Lab) with background subtraction

• Express MKLN1 levels relative to controls using appropriate normalization methods

• Run all comparable samples on the same gel when possible to minimize inter-blot variability

ELISA quantification:

• Generate standard curves using recombinant MKLN1 protein (seven-point curve with 2-fold dilutions)

• Ensure sample measurements fall within the linear portion of the standard curve

• Calculate coefficient of variation (CV) for technical replicates (<10% for intra-assay, <15% for inter-assay)

• Determine limit of detection (LOD) and limit of quantification (LOQ) for your specific assay setup

• Validate dilutional linearity by testing serial dilutions of positive control samples

• Consider using a four-parameter logistic (4PL) curve fit for more accurate quantification

Statistical considerations:

• Perform a minimum of three biological replicates for each experimental condition

• Apply appropriate statistical tests based on data distribution (parametric vs. non-parametric)

• Account for batch effects using appropriate statistical models

• When comparing multiple conditions, apply corrections for multiple testing

• Report both absolute and relative changes in MKLN1 levels where possible

Potential confounding factors:

• MKLN1 subcellular localization changes may affect extraction efficiency

• Post-translational modifications might alter antibody recognition

• Research indicates MKLN1 is a ubiquitin ligase substrate, so proteasome inhibition may affect levels

This methodical approach to quantification ensures reliable measurement of MKLN1 expression levels across experimental conditions.

How do various sample preparation methods affect MKLN1 antibody recognition and signal intensity?

Sample preparation significantly impacts antibody recognition and signal strength when working with MKLN1. Consider these sample preparation variables:

Protein extraction methods comparison:

Fixation impact on epitope accessibility:

• Paraformaldehyde (4%): Preserves most epitopes but may require antigen retrieval

• Methanol fixation: Better for certain cytoskeletal proteins but may alter membrane protein epitopes

• Acetone fixation: Rapid fixation with less cross-linking, good for many cytoplasmic proteins

• Aldehyde-based fixatives: May mask epitopes in the MKLN1 AA 488-614 region, requiring optimization

Antigen retrieval considerations:

• Heat-induced epitope retrieval: Effective for many formalin-fixed samples

• Enzymatic retrieval: Gentler but less predictable results

• pH optimization: Test both acidic (pH 6.0) and basic (pH 9.0) retrieval buffers

• Retrieval time: Balance between sufficient unmasking and potential sample damage

Sample storage impact:

• Fresh vs. frozen samples: Multiple freeze-thaw cycles can degrade MKLN1

• Protease inhibitor importance: Critical for preventing degradation during extraction

• Storage buffer composition: Addition of glycerol (20%) may help preserve protein structure

• Temperature effects: -80°C storage recommended for long-term preservation

When working with MKLN1 antibodies, researchers should systematically optimize sample preparation methods based on their specific application requirements and experimental system to maximize signal sensitivity and specificity.

How can HRP-conjugated MKLN1 antibodies be used to investigate protein-protein interactions in the CTLH complex?

Recent research indicates MKLN1 functions as a substrate of the CTLH ubiquitin ligase complex , opening important avenues for investigation. HRP-conjugated MKLN1 antibodies can be incorporated into several advanced experimental approaches:

Proximity-dependent labeling strategies:

• Employ HRP-conjugated MKLN1 antibodies for proximity-based labeling via tyramide signal amplification (TSA)

• Following immunoprecipitation of MKLN1, use the conjugated HRP to catalyze biotinylation of proximal proteins

• Identify interaction partners through mass spectrometry analysis of biotinylated proteins

• Compare interaction profiles under different cellular conditions (e.g., with/without proteasome inhibition)

Co-immunoprecipitation approaches:

• Use unconjugated MKLN1 antibodies for immunoprecipitation of native complexes

• Detect co-precipitated CTLH complex components using HRP-conjugated antibodies

• Implement sequential immunoprecipitation (tandem IP) to isolate specific subcomplexes

• Compare interaction profiles following MAEA knockout/knockdown, which has been shown to stabilize MKLN1

Functional analysis of ubiquitination:

• Combine MKLN1 immunoprecipitation with ubiquitin-specific antibodies to detect modification status

• Compare ubiquitination patterns in control vs. MAEA-depleted cells

• Analyze how treatments affecting the CTLH complex (e.g., alpelisib) impact MKLN1 stability

• Use cycloheximide chase experiments with MKLN1 antibody detection to determine protein half-life

Visualization of dynamic interactions:

• Employ immunofluorescence with different epitope-targeting antibodies to visualize MKLN1 co-localization with CTLH components

• Implement proximity ligation assay (PLA) using MKLN1 antibodies paired with antibodies against CTLH components

• Analyze how cellular stresses alter the spatial organization of MKLN1 and CTLH components

These approaches leverage the specificity of MKLN1 antibodies to investigate the emerging biology of MKLN1 as a CTLH complex substrate, providing mechanistic insights into its regulation and function.

What are the considerations for using MKLN1 antibodies in studying the relationship between MKLN1 protein and MKLN1-AS in cancer research?

Recent research has identified MKLN1-AS (antisense RNA) as promoting pancreatic cancer progression , suggesting complex regulatory relationships with MKLN1 protein. When investigating these relationships, researchers should consider:

Methodological approaches for protein-RNA relationship studies:

• Implement RNA immunoprecipitation (RIP) using MKLN1 antibodies to identify direct MKLN1-RNA interactions

• Combine with RT-qPCR to specifically detect MKLN1-AS enrichment in immunoprecipitates

• Use UV cross-linking immunoprecipitation (CLIP) for higher-resolution binding site identification

• Compare binding profiles in normoxic vs. hypoxic conditions, given the reported HIF-1α regulation of MKLN1-AS

Expression correlation analysis:

• Develop dual detection systems combining MKLN1 immunohistochemistry with MKLN1-AS RNA in situ hybridization

• Quantify spatial correlation between protein and RNA expression in tissue microarrays

• Analyze expression changes in matched samples following MKLN1-AS modulation

• Correlate expression patterns with clinical outcomes in pancreatic cancer cohorts

Functional investigation strategies:

• Modulate MKLN1-AS levels (overexpression/knockdown) and monitor MKLN1 protein expression using validated antibodies

• Analyze MKLN1 subcellular localization changes in response to MKLN1-AS modulation

• Investigate potential feedback mechanisms where MKLN1 protein might regulate MKLN1-AS expression

• Assess impact of hypoxia on both MKLN1-AS and MKLN1 protein levels simultaneously

Technical considerations:

• Validate antibody specificity in the context of MKLN1-AS manipulation

• Account for potential confounding factors when both are expressed in the same cells

• Design controls to distinguish direct vs. indirect effects on MKLN1 protein expression

• Consider the impact of post-translational modifications on antibody recognition

These approaches integrate antibody-based protein detection with RNA analysis techniques to elucidate the complex interplay between MKLN1 protein and its antisense RNA in cancer contexts.

How can researchers effectively validate the specificity of their MKLN1 antibodies across different experimental models?

Rigorous validation of MKLN1 antibodies is essential for research reproducibility. A comprehensive validation strategy should include:

Cross-species reactivity assessment:

• Test antibody performance across relevant species (human, mouse, rat, zebrafish) based on predicted reactivity

• Evaluate sequence homology at the epitope region (AA 488-614 for HRP-conjugated antibody)

• Validate with recombinant proteins or lysates from multiple species

• Document species-specific banding patterns or signal intensities

Genetic validation approaches:

• Implement CRISPR/Cas9 knockout of MKLN1 as the gold standard for antibody specificity

• Use siRNA or shRNA knockdown with dose-dependent reduction in signal

• Employ heterologous expression systems (overexpression in low/non-expressing cells)

• Compare results from multiple independent genetic perturbation methods

Epitope competition assays:

• Pre-incubate antibody with excess immunizing peptide before application

• Implement gradient competition with increasing peptide concentrations

• Use related peptides to assess cross-reactivity potential

• Compare competition profiles between different MKLN1 antibodies

Multi-method concordance analysis:

• Compare protein detection across different techniques (Western blot, ELISA, immunofluorescence)

• Correlate antibody-based protein detection with mRNA levels

• Verify subcellular localization patterns against known MKLN1 distribution

• Use multiple antibodies targeting different MKLN1 epitopes and compare results

Database integration:

• Document validation results in public repositories (e.g., Antibodypedia)

• Compare observations with published literature on MKLN1 expression patterns

• Assess concordance with proteomics datasets

• Share detailed validation protocols to enhance reproducibility

This systematic validation approach ensures research findings based on MKLN1 antibodies are reliable and reproducible across different experimental models and conditions.

What are the potential applications of MKLN1 antibodies in studying its role in neurodegenerative diseases?

While MKLN1's function in neurodegenerative diseases remains largely unexplored, its kelch-domain structure and potential role in protein degradation pathways suggest promising research directions:

Methodological approaches for neurodegeneration research:

• Implement immunohistochemistry with MKLN1 antibodies on brain tissue from neurodegenerative disease models

• Analyze colocalization with aggregation-prone proteins (e.g., tau, α-synuclein, huntingtin)

• Assess MKLN1 levels and localization in different brain regions affected by neurodegeneration

• Compare expression patterns between control, pre-symptomatic, and disease-stage samples

Functional investigation strategies:

• Analyze MKLN1's potential role in protein quality control via the CTLH complex in neuronal models

• Investigate whether MKLN1 levels correlate with clearance of aggregation-prone proteins

• Assess MKLN1 interaction with neuronal cytoskeletal components using co-immunoprecipitation

• Study MKLN1 dynamics in response to proteotoxic stress in neuronal cultures

Technical considerations:

• Optimize tissue fixation and antigen retrieval for brain-specific applications

• Validate antibody specificity in neural tissues and cell types

• Develop quantitative approaches for analyzing MKLN1 in specific neuronal populations

• Consider post-mortem interval effects on epitope preservation

These approaches could illuminate MKLN1's potential contributions to neuronal homeostasis and protein quality control mechanisms relevant to neurodegenerative disorders.

How might single-cell analysis techniques incorporate MKLN1 antibodies for studying cellular heterogeneity?

Emerging single-cell technologies offer unprecedented opportunities to investigate MKLN1 expression at the individual cell level:

Single-cell protein analysis approaches:

• Adapt MKLN1 antibodies for mass cytometry (CyTOF) by metal conjugation instead of HRP

• Implement imaging mass cytometry to correlate MKLN1 expression with spatial organization in tissues

• Develop antibody panels including MKLN1 and CTLH complex components for multi-parameter analysis

• Optimize signal amplification for detecting low-abundance MKLN1 in single cells

Integrated multi-omics strategies:

• Combine single-cell transcriptomics with antibody-based protein detection (CITE-seq approach)

• Correlate MKLN1 protein and MKLN1-AS RNA at single-cell resolution

• Implement spatial transcriptomics with protein detection to map expression patterns in tissue context

• Analyze cell type-specific MKLN1 expression patterns in complex tissues

Technical optimization considerations:

• Validate antibody specificity at the single-cell level using genetic controls

• Develop fixation and permeabilization protocols compatible with single-cell technologies

• Establish appropriate controls for antibody performance in multiplexed assays

• Implement computational approaches for integrating protein and RNA data at single-cell resolution

These emerging approaches will enable researchers to dissect the heterogeneity of MKLN1 expression and function across diverse cell populations and tissue contexts.

What advances in antibody engineering might enhance future MKLN1 research tools?

Emerging antibody technologies hold promise for developing next-generation MKLN1 research tools:

Novel antibody formats and modifications:

• Development of recombinant antibody fragments (Fab, scFv) targeting MKLN1 with enhanced tissue penetration

• Design of bispecific antibodies targeting MKLN1 and interacting partners simultaneously

• Implementation of pH-sensitive fluorophore conjugates for tracking MKLN1 internalization dynamics

• Creation of conformation-specific antibodies that recognize active vs. inactive MKLN1 states

Advanced conjugation strategies:

• Site-specific conjugation technologies to ensure optimal HRP positioning without affecting binding

• Quantum dot conjugation for long-term imaging with reduced photobleaching

• Click chemistry approaches for modular functionalization of MKLN1 antibodies

• Environmentally-sensitive dye conjugates to detect MKLN1 conformational changes

Intracellular antibody applications:

• Development of cell-permeable MKLN1 antibody formats for live-cell applications

• Creation of intrabodies targeting MKLN1 for real-time visualization of endogenous protein

• Implementation of antibody-based proximity labeling within specific cellular compartments

• Adaptation of nanobody technology for targeting MKLN1 in living cells

Therapeutic potential:

• Exploration of antibody-drug conjugates targeting cancer-specific MKLN1 expression patterns

• Investigation of potential for modulating MKLN1 function or degradation via antibody delivery

• Development of strategies targeting the MKLN1-AS/MKLN1 regulatory axis in pancreatic cancer

These technological advances will expand the toolkit available for MKLN1 research, enabling more sophisticated analyses of its expression, localization, and function in various biological contexts.