MRI1 Antibody

What is the MRI1 protein and why is it important to study?

MRI1 (Methylthioribose-1-phosphate isomerase 1) catalyzes the interconversion of methylthioribose-1-phosphate (MTR-1-P) into methylthioribulose-1-phosphate (MTRu-1-P) . Beyond its enzymatic role, MRI1 has additional functions including promoting cell invasion in response to constitutive RhoA activation by facilitating FAK tyrosine phosphorylation and stress fiber turnover . MRI1 is also known by several alternative names including MRDI, M1Pi, MTR-1-P isomerase, and translation initiation factor eIF-2B subunit alpha/beta/delta-like protein . The protein has a calculated molecular weight of approximately 39 kDa, though it is often observed at 45 kDa in experimental conditions .

What are the common applications for MRI1 antibodies in research?

MRI1 antibodies are primarily utilized in several key applications:

Researchers should note that optimal dilutions are sample-dependent, and titration is recommended for each specific experimental system to obtain optimal results .

How do I determine the optimal antibody concentration for my experiment?

Determining optimal antibody concentration requires systematic titration:

• Start with the manufacturer's recommended concentration range (e.g., 1:500-1:3000 for Western blot)

• Perform a dilution series across this range using your specific samples

• Evaluate signal-to-noise ratio at each concentration

• Consider sample type-specific adjustments (cell lines vs. tissues)

• Test each new batch of antibody before use in critical experiments

What are the best validation approaches to confirm MRI1 antibody specificity?

Comprehensive validation of MRI1 antibody specificity should employ multiple approaches:

• Multi-platform testing: Validate across different applications (WB, IHC, IF) to ensure consistent target recognition

• Knockout/knockdown validation: Compare signal between wild-type and MRI1 knockout/knockdown samples to verify specific binding

• Multi-antibody comparison: Use different antibodies targeting distinct MRI1 epitopes and compare results

• Recombinant protein controls: Test against purified MRI1 protein to confirm binding to the correct target

• Cell-based ELISAs: Test antibody against cells expressing full-length MRI1 protein to verify recognition in cellular context

As exemplified in antibody development protocols: "The primary screen entails parallel ELISAs in two different 96-well plate formats, one employing plates coated with recombinant protein or synthetic peptide immunogen, and the other with plates containing transfected cells expressing the target protein" . This multi-modal approach enhances confidence in antibody specificity.

How should I optimize sample preparation for Western blot analysis with MRI1 antibodies?

For optimal Western blot results with MRI1 antibodies:

• Cell lysis optimization: Use complete lysis buffers containing protease inhibitors to preserve the native 39-45 kDa MRI1 protein

• Sample loading: Load 20-30 μg of total protein per lane for cell lysates; adjust based on MRI1 expression levels

• Control selection: Include positive controls such as HT-1080 or HL-60 cell lysates, which are documented to express detectable MRI1 levels

• Blocking conditions: Use 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Antibody incubation: For primary antibody, incubate at recommended dilution (1:500-1:3000) overnight at 4°C with gentle agitation

• Detection system: HRP-conjugated secondary antibodies with chemiluminescence detection provide appropriate sensitivity for MRI1 detection

What are the recommended protocols for immunohistochemistry with MRI1 antibodies?

For effective IHC with MRI1 antibodies:

• Tissue fixation: Standard 4% formaldehyde fixation is compatible with MRI1 detection

• Antigen retrieval: Use TE buffer pH 9.0 for optimal epitope exposure; citrate buffer pH 6.0 may be used as an alternative

• Antibody dilution: Start with 1:50-1:500 range, optimizing based on tissue type and antibody batch

• Positive control tissue: Rat kidney tissue has shown reliable MRI1 expression and is recommended as a positive control

• Incubation conditions: Optimize incubation time (typically overnight at 4°C) and temperature based on signal intensity

• Detection system: Use appropriate species-specific secondary antibody systems compatible with your visualization method

How can I address weak or absent signal when using MRI1 antibodies?

When facing weak or absent MRI1 signal:

• Antibody validation: Confirm antibody functionality using positive control samples (HT-1080 or HL-60 cells for Western blot; rat kidney for IHC)

• Epitope masking: Consider alternative antigen retrieval methods if standard protocols fail; MRI1 epitopes may be sensitive to different retrieval conditions

• Concentration adjustment: Increase antibody concentration incrementally while monitoring background signal

• Sample handling: Ensure protein degradation is not occurring during sample preparation by adding appropriate protease inhibitors

• Blocking optimization: Test alternative blocking reagents if non-specific binding interferes with detection

• Detection sensitivity: Switch to more sensitive detection systems (e.g., enhanced chemiluminescence substrates for Western blot)

What are common sources of non-specific binding with MRI1 antibodies and how can they be minimized?

To reduce non-specific binding:

• Antibody selection: Choose antibodies validated for specificity; consider those developed using immunoaffinity purification methods

• Blocking optimization: Increase blocking time or concentration; test different blocking agents (milk vs. BSA)

• Wash stringency: Increase number and duration of wash steps with TBST or PBS-T

• Secondary antibody dilution: Ensure secondary antibodies are sufficiently diluted (typically 1:10,000 or higher)

• Cross-reactivity assessment: Be aware that some MRI1 antibodies may cross-react with related proteins; validate using knockout controls when possible

• Sample complexity: Consider pre-clearing complex samples if high background persists

How stable are MRI1 antibodies and what are the best storage practices?

For optimal MRI1 antibody stability:

• Storage temperature: Store at -20°C for long-term storage; many commercial MRI1 antibodies remain stable for up to one year under these conditions

• Aliquoting: For 100μl size antibodies, aliquoting is often unnecessary for -20°C storage, but may be beneficial for larger volumes

• Freeze-thaw cycles: Minimize freeze-thaw cycles; repeated cycles can lead to antibody degradation and reduced activity

• Working dilutions: Store diluted working solutions at 4°C for short-term use (up to one month)

• Buffer composition: MRI1 antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which enhances stability

• Quality control: Test antibody performance periodically, especially before critical experiments

How can MRI1 antibodies be effectively used in multiplexed immunoassays?

For multiplexed detection involving MRI1:

• Isotype selection: Utilize MRI1 antibodies of non-IgG1 subclasses to facilitate multiplex labeling with subclass-specific secondary antibodies

• Species differentiation: Combine rabbit-derived MRI1 antibodies with mouse antibodies targeting other proteins to enable species-specific detection

• Spectral separation: When using fluorescent detection methods, ensure adequate spectral separation between fluorophores

• Sequential staining: For challenging multiplex applications, consider sequential rather than simultaneous staining protocols

• Cross-reactivity testing: Validate all antibodies in the multiplex panel individually before combining to ensure no cross-reactivity

• Internal controls: Include internal controls for co-localization or mutual exclusivity as appropriate to your experimental design

For example, research protocols note: "We also describe the special attention given to candidates with less common non-IgG1 IgG subclasses that can facilitate simultaneous multiplex labeling with subclass-specific secondary antibodies" .

What considerations are important when using MRI1 antibodies across different species?

For cross-species MRI1 detection:

• Sequence homology: Verify antibody epitope conservation across target species; MRI1 shows high sequence homology across human, mouse, and rat

• Validated reactivity: Most commercial MRI1 antibodies have been validated for human, mouse, and rat reactivity; some also work in additional species including rabbit, dog, cow, pig, guinea pig, and monkey

• Species-specific controls: Include appropriate positive and negative controls from each target species

• Epitope mapping: Consider the specific epitope region; antibodies targeting amino acids 94-143 of human MRI1 show broad cross-species reactivity due to high sequence conservation

• Dilution adjustments: Optimize antibody dilutions separately for each species as expression levels and background may vary

As noted in one antibody validation study: "Based on the high level of identity among human, mouse, and rat MRP1 protein sequence, we produced a specific polyclonal antibody against a synthetic polypeptide covering the C-terminus of the human protein" which successfully detected the target across species . Similar approaches have been applied to MRI1 antibody development.

How can MRI1 antibodies be used to investigate protein-protein interactions and signaling pathways?

For studying MRI1's role in protein interactions:

• Co-immunoprecipitation: Use MRI1 antibodies to pull down protein complexes, followed by analysis of binding partners

• Proximity ligation assays: Combine MRI1 antibodies with antibodies against suspected interaction partners to visualize protein proximity in situ

• Chromatin immunoprecipitation: If investigating potential DNA interactions, optimize ChIP protocols with MRI1 antibodies

• Phosphorylation analysis: Combine with phospho-specific antibodies to investigate MRI1's role in FAK tyrosine phosphorylation and stress fiber turnover

• Functional blocking: Test if antibodies binding specific MRI1 domains affect its enzymatic activity or interaction capabilities

• Domain-specific antibodies: Utilize antibodies targeting different regions of MRI1 to investigate domain-specific interactions

Understanding these protein interactions is particularly relevant given that "Independently from catalytic activity, [MRI1] promotes cell invasion in response to constitutive RhoA activation by promoting FAK tyrosine phosphorylation and stress fiber turnover" .

How are patient-centric sampling approaches changing the landscape of antibody-based diagnostics, including those for MRI1?

Patient-centric sampling is transforming antibody-based research:

• Microsampling technologies: Volume absorptive microsampling (VAMS) with plastic substrates allows for smaller sample volumes (as little as 20 μL) while maintaining analytical integrity

• Stability advances: Samples collected via microsampling methods can remain stable for extended periods (at least 6 months at room temperature), facilitating field collection and storage

• Bridging studies: Clinical validation studies demonstrate equivalence between traditional venipuncture and microsampling methodologies, showing "fully matched profiles for venous serum vs. capillary blood VAMS"

• Ethical advantages: Reduced blood volume requirements are particularly beneficial for pediatric studies and longitudinal sampling protocols

• Compartment considerations: When analyzing antibody concentrations from different sample types, researchers must account for compartment-specific partitioning effects

These advances are exemplified in current clinical programs: "Ethical Benefits: Obtaining samples from infants, collection of samples in a closer timeframe to a clinical event, freeing-up blood volume to collect additional samples. Improved Patient Experience: Sample collection in settings more convenient to the patient, limiting disruption to normal life for clinical study subjects, less invasive than venipuncture" .

What are the latest advances in antibody validation technologies that could improve MRI1 detection specificity?

Recent validation technologies enhancing antibody specificity include:

• CRISPR/Cas9 validation: Generation of knockout cell lines specifically for antibody validation provides definitive negative controls

• Orthogonal validation: Comparison of antibody-based results with orthogonal methods (mass spectrometry, RNA-seq) to confirm target specificity

• Automated high-throughput screening: Development of systematic multi-step mAb screening focused on identifying antibodies with efficacy and specificity in labeling specific sample types

• Subclass-specific validation: Enhanced screening for less common non-IgG1 IgG subclasses facilitates multiplex labeling applications

• Cell-based ELISA refinements: Improved screening against transiently transfected cells expressing full-length target protein under conditions mimicking intended applications

As described in advanced validation protocols: "We provide examples from NeuroMab screens, and from the subsequent specialized validation of those selected as NeuroMabs. We highlight the particular challenges and considerations of determining specificity for brain immunolabeling" . Similar comprehensive validation approaches are being applied to antibodies for various targets including MRI1.

How might complementary methodologies optimize the analysis of MRI1 in complex biological samples?

Optimizing MRI1 analysis through complementary methods:

• Size exclusion chromatography (SEC) paired with immunodetection: Enables analysis of MRI1 aggregation states prior to antibody detection

• Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) integration: Provides enhanced resolution of protein heterogeneity when standard Western blot analysis is insufficient

• Mass spectrometry validation: Orthogonal confirmation of antibody-detected MRI1 through peptide mass fingerprinting

• Machine learning algorithms: Application of pattern recognition to complex immunohistochemical datasets to identify subtle differences in MRI1 expression patterns

• Dynamic MRI lesion analysis: In neurological contexts, correlation of MRI lesion dynamics with molecular markers like MRI1 can provide insights into disease progression

These complementary approaches address the understanding that "Size heterogeneity is a critical quality attribute (CQA)... as both aggregation and degradation can impact the safety and efficacy..." . While this principle is described for therapeutic antibodies, it applies equally to the detection and characterization of target proteins like MRI1.