Recombinant Uncharacterized protein Rv1945/MT1995 (Rv1945, MT1995)

What is Rv1945/MT1995 protein and what organism does it originate from?

Rv1945/MT1995 is an uncharacterized protein from Mycobacterium tuberculosis. According to the available data, it has a UniProt ID of P95269 and consists of 454 amino acids in its full-length form . As an uncharacterized protein, its specific biological role in M. tuberculosis pathogenesis remains to be fully elucidated, making it an important target for tuberculosis research.

What are the optimal storage and reconstitution protocols for Rv1945/MT1995 recombinant protein?

For optimal experimental outcomes, researchers should follow these evidence-based protocols:

Storage conditions:

• Store lyophilized protein at -20°C/-80°C for up to 12 months

• Store reconstituted protein in aliquots at -20°C/-80°C for up to 6 months

• Working aliquots can be stored at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as this may compromise protein integrity

Reconstitution protocol:

• Briefly centrifuge the lyophilized product before opening to bring contents to the bottom

• Reconstitute the protein to a concentration of 0.1-1.0 mg/mL using deionized sterile water

• Add glycerol to a final concentration of 5-50% (the default reference concentration is 50%)

• Prepare multiple aliquots for long-term storage to minimize freeze-thaw cycles

What expression systems are utilized for Rv1945/MT1995 recombinant protein production?

The available data indicates that Rv1945/MT1995 recombinant protein is typically produced using mammalian cell expression systems . This expression system is often preferred for proteins requiring complex folding or post-translational modifications to maintain their native structure and function.

When planning to work with this protein, researchers should consider:

• The expression system's impact on protein folding and modifications

• Whether the protein format (lyophilized powder) is suitable for intended applications

• The recommended application fields (Western Blot, ELISA) mentioned in product specifications

How can researchers design effective experiments to characterize the function of Rv1945/MT1995?

When designing experiments to characterize this uncharacterized protein, researchers should implement a systematic approach:

• Sequence-based functional prediction:

  • Perform bioinformatic analysis to identify conserved domains and motifs

  • Compare with characterized proteins from related mycobacterial species

  • Use computational tools to predict potential functions

• Experimental design considerations:

  • Incorporate appropriate controls including positive controls (known M. tuberculosis proteins) and negative controls

  • Design experiments with sufficient statistical power based on preliminary studies

  • Account for inter-individual variability in experimental systems, as this can significantly impact results

• Validation strategies:

  • Use multiple complementary techniques to confirm findings

  • Implement a multi-dimensional approach similar to those used in single-cell studies of microbes

  • Consider both in vitro and cellular systems to validate functional hypotheses

What methodological considerations should be taken when optimizing assays with Rv1945/MT1995?

Optimizing assays with Rv1945/MT1995 requires several methodological considerations:

• Buffer optimization:

  • Test multiple buffer conditions (Tris/PBS-based buffers at pH 8.0 are mentioned in product information)

  • Evaluate the effect of additives like trehalose (6% is mentioned in storage buffer)

  • Determine optimal protein concentration ranges for specific assay types

• Assay development strategy:

  • Begin with well-established assays (WB, ELISA) that are recommended for this protein

  • Implement systematic parameter optimization similar to approaches used in model-based meta-analysis (MBMA)

  • Document all optimization steps to establish reproducible protocols

• Quality control measures:

  • Verify protein integrity before each experiment (SDS-PAGE is mentioned for purity assessment)

  • Include stability controls to account for potential degradation during experimental procedures

  • Implement positive and negative controls to validate assay performance

How should researchers incorporate inter-individual variability when designing experiments with Rv1945/MT1995?

Research has demonstrated that accounting for inter-individual variability significantly improves the quality of experimental results. When working with Rv1945/MT1995:

• Sample size determination:

  • Conduct preliminary studies to assess variability in your experimental system

  • Use statistical power calculations to determine appropriate sample sizes

  • Consider stratified analysis approaches if distinct response patterns emerge

• Experimental design strategies:

  • Implement clustering procedures to identify multidimensional response types, as demonstrated in mouse studies

  • Systematically incorporate individual response types in the experimental design

  • Consider repeated measures designs to capture within-subject variability

• Data analysis approaches:

  • Use multivariate analysis methods to account for complex response patterns

  • Implement mixed-effects models to separate inter-individual from intra-individual variability

  • Consider Bayesian approaches for integrating prior knowledge with experimental data

As demonstrated in published research, failure to account for inter-individual variability can lead to misinterpretation of results in pharmacological experiments .

What approaches can be used to validate the specificity of interactions involving Rv1945/MT1995?

To ensure the specificity of interactions involving Rv1945/MT1995, researchers should implement a multi-faceted validation strategy:

• Biochemical validation:

  • Use tagged versions of the protein (tags may be present on either N-terminal or C-terminal depending on stability requirements)

  • Perform competition assays with excess unlabeled protein

  • Implement dose-response studies to confirm specific binding relationships

• Structural validation:

  • Generate truncation mutants to map interaction domains

  • Use site-directed mutagenesis to identify critical residues

  • Apply techniques like Raman-FISH that enable direct visualization of specific interactions

• Controls and counter-screens:

  • Include structurally related but functionally distinct proteins as controls

  • Perform parallel assays under varying conditions to distinguish specific from non-specific interactions

  • Validate findings across multiple experimental platforms and conditions

How can researchers integrate Rv1945/MT1995 studies into broader tuberculosis research?

Integrating Rv1945/MT1995 research into the broader tuberculosis research landscape requires strategic approaches:

• Contextual experimental design:

  • Design experiments that connect Rv1945/MT1995 to known tuberculosis pathogenesis pathways

  • Investigate potential interactions with host immune factors

  • Consider the protein's role in various phases of the bacterial life cycle

• Translational considerations:

  • Apply forward and reverse translational approaches similar to those used in vaccine and antibody development

  • Develop mathematical models that integrate experimental data across different studies

  • Consider how findings might contribute to diagnostic or therapeutic applications

• Collaborative research frameworks:

  • Implement interdisciplinary approaches combining structural biology, functional genomics, and immunology

  • Establish standardized protocols to enable cross-laboratory validation

  • Contribute to open-access databases for M. tuberculosis protein characterization

What novel methodologies might be particularly valuable for studying uncharacterized proteins like Rv1945/MT1995?

Emerging methodologies offer new opportunities for characterizing proteins like Rv1945/MT1995:

• Single-cell approaches:

  • Apply single-cell methods that have been effectively used to study aquatic microbes

  • Implement Raman spectroscopy combined with fluorescence in situ hybridization (Raman-FISH) to study protein localization and interaction dynamics

  • Use CRM (confocal Raman microscopy) to identify specific microbial taxa while simultaneously obtaining spectral data

• Computational prediction integration:

  • Implement integrated computational-experimental workflows

  • Apply AlphaFold or similar AI tools for structure prediction to guide experimental design

  • Develop custom machine learning approaches trained on mycobacterial protein datasets

• Systems biology frameworks:

  • Design experiments within a systems biology framework to place Rv1945/MT1995 in its biological context

  • Apply network analysis to identify potential functional relationships

  • Implement multi-omics approaches to comprehensively characterize the protein's role