MIOS Antibody

What is MIOS protein and what cellular functions does it perform?

MIOS (Missing Oocyte Meiosis Regulator Homolog) is a 99 kDa protein that functions as a component of the GATOR2 complex. It plays critical roles in:

• Activating the amino acid-sensing branch of the mTORC1 signaling pathway

• Indirectly activating mTORC1 through inhibition of the GATOR1 subcomplex

• Contributing to E3 ubiquitin-protein ligase activity toward GATOR1

• Preventing autoubiquitination of WDR24, the catalytic subunit of the GATOR2 complex

• Supporting brain myelination (based on similarity studies)

Within the cellular context, MIOS contains six N-terminal WD40 repeats and a C-terminal zinc finger-like domain. Its structural features contribute to its protein-protein interaction capabilities within the GATOR2 complex .

What types of MIOS antibodies are available for research applications?

Researchers can access several types of MIOS antibodies:

When selecting an antibody, consider your experimental requirements including application type, species reactivity, and whether epitope specificity or broader recognition is more important for your research aims .

What is the role of MIOS in the mTORC1 signaling pathway?

MIOS participates in a complex regulatory network controlling mTORC1 activity:

• As a GATOR2 component, MIOS acts as an activator of the amino acid-sensing branch of mTORC1 signaling

• In amino acid abundance, the GATOR2 complex (containing MIOS) mediates ubiquitination of NPRL2, a core component of the GATOR1 complex, leading to GATOR1 inactivation

• This inactivation of GATOR1 releases its inhibitory effect on mTORC1, allowing mTORC1 activation

• Conversely, in amino acid deprivation, GATOR2 is inhibited, which activates GATOR1, ultimately leading to mTORC1 inhibition

• Within the GATOR2 complex, MIOS specifically prevents autoubiquitination of WDR24, maintaining the structural integrity of the complex

Understanding these interactions is crucial when designing experiments targeting or utilizing MIOS for mTORC1 pathway investigations.

What are the optimal conditions for Western blot detection of MIOS?

For effective Western blot detection of MIOS protein:

• Sample preparation: Use cell lines with confirmed MIOS expression such as HeLa, 293T, Jurkat, MCF-7, or K-562 cells for human samples; TCMK-1 or NIH 3T3 for mouse samples; and Rat-2 or rat liver tissue for rat samples

• Antibody dilutions:

  • For polyclonal antibodies: Use 1:500-1:1000 dilution for primary antibody

  • For monoclonal antibodies: Optimal concentration is 0.1-2 μg/mL depending on the specific antibody

• Detection methods: Use chemiluminescence with exposure times ranging from 30 seconds to 3 minutes depending on signal strength

• Running conditions: MIOS has a predicted molecular weight of 99 kDa; use reducing conditions and appropriate percentage gels (typically 8-10%) to effectively resolve this protein

Success has been demonstrated using PVDF membranes with appropriate blocking and washing steps, though specific protocols may need optimization for your particular antibody and experimental system .

How can I validate the specificity of a MIOS antibody for my research applications?

Validating MIOS antibody specificity requires a multi-approach strategy:

• Positive controls: Include lysates from cell lines known to express MIOS (HeLa, 293T, Jurkat, MCF-7 for human; TCMK-1, NIH 3T3 for mouse; Rat-2 for rat)

• Molecular weight verification: Confirm detection at the expected molecular weight (~99 kDa)

• Knockdown/knockout validation: Use siRNA or CRISPR to generate MIOS-depleted cells as negative controls. Published studies have used this approach to validate MIOS antibodies

• Cross-reactivity testing: If working across species, test the antibody on samples from each species to confirm cross-reactivity matches manufacturer claims

• Immunoprecipitation verification: For antibodies used in IP experiments, confirm by subsequent Western blot analysis. For example, when using ab202274 for IP of 293T whole cell lysates (1 mg for IP, 20% of IP loaded) at 6 μg/mg lysate, followed by Western blot at 0.4 μg/ml, specific bands were observed, while control IgG IP showed no signal

This comprehensive validation approach ensures confidence in experimental results and minimizes the risk of non-specific binding artifacts.

What are the recommended immunoprecipitation protocols for MIOS protein research?

For successful immunoprecipitation of MIOS:

• Sample preparation:

  • Use 1.0-3.0 mg of total protein lysate from appropriate cell lines (HeLa, 293T cells show good results)

  • Ensure complete cell lysis using appropriate buffers containing protease inhibitors

• Antibody requirements:

  • For polyclonal antibodies: Use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate

  • For ab202274: Use at 6 μg/mg lysate for IP

• Protocol overview:

  • Prepare cell lysate in appropriate lysis buffer

  • Pre-clear with protein A/G beads if background is a concern

  • Add recommended amount of MIOS antibody to lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add protein A/G beads and incubate for 1-4 hours

  • Wash beads thoroughly (3-5 times) with wash buffer

  • Elute proteins by boiling in sample buffer

  • Analyze by Western blot (load approximately 20% of IP material)

• Controls: Always include a control IgG IP from the same species as your MIOS antibody to identify non-specific binding

This protocol has been validated in multiple studies and can be optimized for specific experimental conditions.

How can I address weak or absent signal when using MIOS antibodies in Western blotting?

When experiencing weak or absent MIOS signals in Western blotting:

• Protein expression verification:

  • Confirm MIOS expression in your cell line/tissue using published data

  • Consider that expression levels may vary across cell types and conditions

• Sample preparation optimization:

  • Increase protein concentration (load 50-100 μg total protein)

  • Use fresh lysates when possible

  • Ensure complete denaturation by adequate heating in sample buffer

  • Add protease inhibitors to prevent degradation

• Antibody-specific adjustments:

  • Increase antibody concentration (try 2-fold higher concentration)

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

  • Optimize blocking conditions (try different blockers: BSA vs. milk)

  • For polyclonal antibodies, consider longer exposure times (up to 3 minutes)

• Detection enhancements:

  • Use high-sensitivity ECL substrates

  • Try longer exposure times

  • Consider signal amplification methods

• Epitope accessibility issues:

  • If using a specific domain-targeted antibody (e.g., antibodies targeting aa 600-750 or 800 to C-terminus regions), consider that protein modifications or interactions might mask the epitope

Systematic troubleshooting through these parameters should help identify and resolve detection issues.

What are the potential cross-reactivity concerns with MIOS antibodies and how can they be addressed?

Managing cross-reactivity with MIOS antibodies requires careful analysis and controls:

• Sources of cross-reactivity:

  • Structural homology between MIOS and related proteins

  • Conservation across species (human MIOS shares 98% identity with mouse and rat MIOS over aa 620-875)

  • Non-specific binding due to antibody concentration or blocking issues

• Identification strategies:

  • Run validated positive controls alongside experimental samples

  • Include samples from MIOS knockdown/knockout cells

  • Look for unexpected bands at molecular weights different from the predicted 99 kDa

• Resolution approaches:

  • Titrate antibody to find optimal concentration that maximizes specific binding while minimizing non-specific signals

  • Increase stringency of washing steps

  • Use more selective blocking agents

  • Consider alternative antibodies targeting different epitopes

  • For critical experiments, validate findings with two different MIOS antibodies

• Species considerations:

  • When working across species, verify the degree of homology in the epitope region

  • Remember that while human MIOS shares high homology with mouse and rat in some regions, other regions may be more divergent, affecting antibody performance

Establishing these controls and optimization steps ensures greater confidence in experimental outcomes when using MIOS antibodies.

How should I interpret variations in MIOS detection between different cell lines or tissues?

When analyzing variations in MIOS detection across different biological samples:

• Biological expression differences:

  • MIOS expression likely varies naturally between cell types and tissues based on metabolic demands and mTORC1 pathway activity

  • Consider the cell's metabolic state, as amino acid sensing (a key function of MIOS) may affect expression or localization

• Technical considerations:

  • Extraction efficiency may vary between cell types or tissues due to different cellular matrices

  • Protein-protein interactions in specific cell types might mask epitopes

  • Post-translational modifications may differ between cell types, affecting antibody recognition

• Analytical approach:

  • Use loading controls appropriate for your sample types

  • Normalize MIOS signal to total protein rather than single housekeeping proteins when comparing across diverse tissues

  • Consider using multiple antibodies targeting different MIOS epitopes for confirmation

  • Verify with orthogonal methods (qPCR for mRNA levels, mass spectrometry)

• Documented variations:

  • MIOS has been successfully detected in various human cell lines including HeLa, 293T, Jurkat, MCF-7, K-562, and PC-3

  • In mouse, detection has been confirmed in TCMK-1, NIH 3T3, and 3T3-L1 cell lines

  • In rat, detection has been verified in Rat-2 cell line and rat liver tissue

Understanding both biological and technical sources of variation will lead to more accurate interpretation of MIOS expression data.

How can MIOS antibodies be utilized in studying the mTORC1 signaling pathway and amino acid sensing?

MIOS antibodies serve as valuable tools for investigating mTORC1 regulation:

• Pathway interaction studies:

  • Use MIOS antibodies in co-immunoprecipitation to investigate interactions with other GATOR complex components

  • Combine with antibodies against GATOR1 components (DEPDC5, NPRL2, NPRL3) to study complex formation and dissociation

  • Investigate MIOS interactions with SESTRIN2, which is involved in amino acid sensing upstream of mTORC1

• Nutrient response experiments:

  • Monitor MIOS localization and complex formation during amino acid starvation and refeeding

  • Combine with phospho-specific antibodies against mTORC1 substrates (p-S6K, p-4EBP1) to correlate MIOS status with pathway activity

• Ubiquitination studies:

  • Use MIOS antibodies alongside ubiquitin antibodies to study how GATOR2 mediates ubiquitination of NPRL2

  • Investigate how MIOS prevents autoubiquitination of WDR24

• Functional validation:

  • Supplement antibody-based detection with MIOS knockdown/knockout approaches

  • Compare phenotypes of MIOS depletion with inhibition of mTORC1 (rapamycin treatment)

  • Assess effects on downstream processes like protein synthesis, autophagy, and cell growth

These applications leverage MIOS antibodies to dissect the complex regulatory mechanisms governing cellular nutrient sensing and growth control.

What considerations are important when using MIOS antibodies in super-resolution imaging techniques?

When employing MIOS antibodies for super-resolution microscopy:

• Antibody selection criteria:

  • Choose antibodies validated for immunofluorescence applications

  • Consider using directly labeled primary antibodies to minimize localization error

  • Monoclonal antibodies may provide more precise localization than polyclonals

• Sample preparation optimization:

  • Use appropriate fixation methods that preserve protein localization while maintaining epitope accessibility

  • Consider epitope retrieval methods if necessary (PBS-based buffers are commonly used)

  • Optimize permeabilization to balance antibody access with preservation of cellular structures

• Advanced imaging techniques:

  • For techniques like immuno-OligoSTORM (iOS), which combines OligoSTORM and DNA-PAINT imaging, careful antibody selection is critical

  • This approach allows simultaneous super-resolved imaging of DNA at specific gene loci and associated proteins like MIOS

• Multi-protein imaging considerations:

  • When studying MIOS alongside other GATOR2 complex components (WDR24, WDR59, SEH1L, SEC13), ensure antibodies are compatible for multi-color imaging

  • Consider cross-reactivity issues and appropriate controls when imaging multiple proteins

• Data interpretation:

  • Reconcile single-cell super-resolution data with population-based biochemical data

  • Consider that MiOS (Modeling immuno-OligoSTORM) approaches can help integrate imaging and computational strategies to model 3D structures while accounting for single-cell variability

Super-resolution techniques provide valuable insights into MIOS localization and interactions at a resolution unattainable with conventional microscopy.

What are the best practices for quantifying MIOS protein levels in comparative studies?

For accurate quantification of MIOS across experimental conditions:

• Sample preparation standardization:

  • Use consistent lysis buffers and protein extraction protocols

  • Determine protein concentration using reliable methods (BCA or Bradford assays)

  • Load equal amounts of total protein per lane (typically 50 μg)

• Western blot quantification:

  • Use gradient gels for better resolution of the 99 kDa MIOS protein

  • Include multiple technical replicates

  • Use housekeeping proteins appropriate for your experimental conditions (β-actin, GAPDH, tubulin)

  • Consider using total protein normalization methods (Stain-Free technology, Ponceau staining)

• Experimental design considerations:

  • Include a standard curve using recombinant MIOS or serially diluted positive control lysate

  • For comparative studies, process all samples in parallel to minimize inter-blot variation

  • When comparing effects of treatments (e.g., amino acid starvation), include appropriate time-matched controls

• Image acquisition and analysis:

  • Use a digital imaging system with a linear dynamic range

  • Avoid saturated signals by optimizing exposure times

  • Use analysis software that allows background subtraction

  • For densitometry, define consistent region of interest areas

• Statistical considerations:

  • Perform at least three biological replicates

  • Apply appropriate statistical tests based on your experimental design

  • Report data with measures of dispersion (standard deviation or standard error)

Following these quantification practices ensures reliable comparative data when measuring MIOS protein levels across different experimental conditions.

How can MIOS antibodies be utilized in studying neurodevelopmental processes and myelination?

MIOS antibodies offer valuable tools for investigating neural development:

• Brain myelination studies:

  • MIOS and the GATOR2 complex are implicated in brain myelination processes

  • Use immunohistochemistry with MIOS antibodies on brain tissue sections to examine expression patterns during developmental stages

  • Compare MIOS localization with myelin markers to establish temporal relationships

• Cell-type specific analysis:

  • Combine MIOS antibody staining with cell-type specific markers (oligodendrocytes, neurons, astrocytes) to determine which neural cells express MIOS

  • Use fluorescence-activated cell sorting (FACS) with MIOS antibodies to isolate specific neural cell populations for further analysis

• Functional investigations:

  • Examine effects of MIOS knockdown/knockout on oligodendrocyte differentiation and myelination

  • Investigate potential interactions between MIOS/GATOR2 and signaling pathways known to regulate myelination

  • Study how amino acid sensing through MIOS might regulate protein synthesis required for myelin production

• Technical considerations:

  • For brain tissue, suggested antigen retrieval with TE buffer pH 9.0 has shown good results, though citrate buffer pH 6.0 is an alternative option

  • When working with tissue sections, optimize antibody concentration (starting with 1:50-1:500 dilution range)

This application area represents an emerging field connecting nutrient sensing pathways with neurodevelopmental processes through MIOS and the GATOR complexes.

What role can MIOS antibodies play in investigating metabolic disorders related to mTORC1 dysregulation?

MIOS antibodies can provide insights into metabolic disease mechanisms:

• Diabetes and insulin resistance studies:

  • Examine MIOS expression and complex formation in insulin-responsive tissues (muscle, liver, adipose)

  • Investigate correlations between MIOS levels and insulin signaling markers

  • Compare MIOS-GATOR2 complex integrity in normal versus diabetic models

• Cancer metabolism research:

  • Analyze MIOS expression across cancer cell lines and tumor samples

  • Investigate MIOS involvement in cancer-associated mTORC1 hyperactivation

  • Use MIOS antibodies to assess response to mTORC1-targeted therapies

• Aging and cellular senescence:

  • Explore age-related changes in MIOS expression and function

  • Investigate connections between MIOS/GATOR2 and cellular senescence pathways

  • Use MIOS antibodies to track changes in nutrient sensing capacity during aging

• Technical approaches:

  • Tissue microarrays with MIOS antibodies can efficiently screen multiple samples

  • Combine with phospho-specific antibodies against mTORC1 substrates to correlate MIOS status with pathway activity

  • Consider measuring ubiquitination status of GATOR1 components as a readout of MIOS/GATOR2 activity

• Translation to therapy:

  • Using MIOS antibodies to evaluate potential therapeutic strategies targeting the GATOR complexes

  • Screening compounds that might modulate MIOS-dependent mTORC1 regulation

  • Developing biomarkers for patient stratification based on MIOS/GATOR2 status

MIOS antibody-based research may uncover novel targets for metabolic disorders where mTORC1 dysregulation plays a central role.