Recombinant Mouse Transmembrane protein 88B (Tmem88b)

What is Tmem88b and what are its basic characteristics?

Tmem88b (transmembrane protein 88B) is a protein-coding gene located on chromosome 4 in mice (Mus musculus). The protein contains 173 amino acids and is predicted to be localized in the cell membrane . The official full name is "transmembrane protein 88B" with gene ID 320587 . Tmem88b belongs to the transmembrane protein 88 family (IPR033355) and shares structural similarities with its paralog Tmem88. The protein contains transmembrane domains that anchor it within the cell membrane, with portions extending into both intracellular and extracellular spaces. According to structural predictions, Tmem88b features hydrophobic segments consistent with its transmembrane nature .

How is Tmem88b different from Tmem88?

Tmem88b is a paralog of Tmem88, meaning they are related by gene duplication events within the mouse genome. While both belong to the same protein family (transmembrane protein 88), they exhibit distinct expression patterns and potentially different functions . Tmem88 (encoded by gene ID 67020) has been more extensively studied than Tmem88b . Sequence alignment reveals approximately 60-70% similarity between the two proteins, with conservation primarily in the transmembrane domains and certain functional motifs. The divergence between these paralogs suggests possible specialization of function, though more research is needed to fully characterize their distinct roles .

What is known about the biological function of Tmem88b?

The biological function of Tmem88b is still being elucidated, but based on current research and orthologous relationships:

• Tmem88b is predicted to be involved in negative regulation of the canonical Wnt signaling pathway

• It is predicted to be active in the plasma membrane

• It may have roles in developmental processes, as indicated by expression studies in zebrafish

• Its paralog Tmem88 has been better characterized functionally, providing clues to potential shared functions

Further studies, particularly those using knockout models or CRISPR-mediated targeting (as mentioned in search result #2), are needed to fully clarify its physiological role. Recent research has suggested potential connections to neuronal function, as indicated by transcriptomic studies of the medial prefrontal cortex (mPFC) .

Expression and Purification Methods

For recombinant Tmem88b purification, consider the following strategies based on the protein's characteristics and tags:

• Affinity Chromatography:

  • For His-tagged Tmem88b: Use immobilized metal affinity chromatography (IMAC) with Ni-NTA or Co2+ matrices

  • For Fc-tagged constructs: Protein A or Protein G columns are effective

  • For Avi-tagged proteins: Avidin or streptavidin-based purification systems

• Additional Purification Steps:

  • Size exclusion chromatography to achieve >80% purity

  • Ion exchange chromatography as a polishing step

• Detergent Considerations:

  • As a transmembrane protein, inclusion of mild detergents (0.03-0.1% DDM or 0.1-0.5% CHAPS) during purification is crucial to maintain solubility and native conformation

  • Consider detergent exchange during purification process

• Quality Control:

  • Verify purity using SDS-PAGE and Western blot analysis

  • Assess endotoxin levels (should be <1.0 EU per μg protein)

For optimal results, the protein should be stored in PBS buffer, either as a liquid at 4°C for short-term storage or lyophilized at -20°C to -80°C for long-term storage .

How can I use recombinant Tmem88b in signaling pathway studies?

To investigate Tmem88b's role in signaling pathways, particularly the Wnt signaling pathway, consider these methodological approaches:

• Wnt Signaling Analysis:

  • Use TOPFlash/FOPFlash luciferase reporter assays to measure changes in canonical Wnt pathway activity when recombinant Tmem88b is introduced to cells

  • Co-immunoprecipitation experiments to identify binding partners within the Wnt pathway

  • Compare signaling outputs between wild-type cells and those overexpressing or lacking Tmem88b

• Protein-Protein Interaction Studies:

  • Proximity ligation assays to detect interactions with suspected binding partners in situ

  • Pull-down assays using the recombinant protein as bait

  • FRET-based approaches for real-time monitoring of interactions

• Functional Assays:

  • Phosphorylation assays to monitor downstream effects on signaling cascades

  • Cellular localization studies using fluorescently tagged Tmem88b

  • Competition assays with the related Tmem88 protein to assess functional overlap

When designing these experiments, carefully control for tag effects by including appropriate controls and consider using carrier-free preparations for sensitive signaling assays to avoid BSA interference .

What protocols should be followed for using Tmem88b in ELISA-based detection?

For ELISA-based detection of Tmem88b, follow these methodological guidelines:

• Sample Preparation:

  • For tissue homogenates: Use a buffer containing mild detergent (0.1% Triton X-100 or NP-40) with protease inhibitors

  • For cell lysates: Lyse cells in appropriate buffer (RIPA or NP-40 buffer with protease inhibitors)

  • Centrifuge samples (10,000 × g for 10 minutes) to remove debris

• Assay Protocol:

  • Use commercial kits with a detection range of 0.156-10 ng/ml for optimal sensitivity

  • Prepare standards using recombinant Tmem88b (preferably from the same species)

  • Perform all incubations at room temperature unless specified otherwise

  • Include technical replicates (minimum triplicate) for all samples

• Data Analysis:

  • Generate a standard curve using 4- or 5-parameter logistic regression

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

  • Calculate intra- and inter-assay coefficients of variation to assess reproducibility

• Critical Considerations:

  • Be aware that ELISA kits are optimized for native proteins; detection of recombinant proteins may vary

  • Sample dilution is crucial - aim for mid-range kit detection (0.5-5 ng/ml)

  • Validate antibody specificity using positive and negative controls

  • Consider matrix effects that might interfere with detection

How can I use CRISPR techniques to study Tmem88b function?

For CRISPR-based functional studies of Tmem88b, follow these methodological approaches:

• Guide RNA Design and Selection:

  • Use validated gRNA sequences designed to specifically target the Tmem88b gene with minimal off-target effects

  • When targeting Tmem88b, consider ordering at least two gRNA constructs to increase success probability

  • Double-check gRNA sequences against your specific mouse strain sequence, as genomic variations may affect targeting efficiency

• Experimental Design for Knockout Studies:

  • For full gene knockout: Target early exons or multiple exons simultaneously

  • For specific domain analysis: Design gRNAs to create in-frame deletions of specific domains

  • For paralog comparison: Design parallel knockouts of both Tmem88 and Tmem88b

• Delivery Methods:

  • For cell lines: Transfection or viral delivery of CRISPR components

  • For in vivo studies: Consider viral delivery or pronuclear injection for germline modification

  • For temporal control: Use inducible CRISPR systems (e.g., Tet-regulated Cas9)

• Validation Approaches:

  • Genomic validation: PCR amplification and sequencing of the target region

  • Protein validation: Western blot or immunostaining to confirm protein loss

  • Functional validation: Assess changes in Wnt signaling or other suspected pathways

• Controls and Considerations:

  • Include non-targeting gRNA controls

  • Address potential compensatory upregulation of the paralog Tmem88

  • Design rescue experiments using the recombinant Tmem88b protein or expression constructs

When analyzing results, consider the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules for regulatory compliance .

What is the relationship between Tmem88b and Wnt signaling regulation?

The relationship between Tmem88b and Wnt signaling is an emerging area of research, built upon evidence from its paralog Tmem88 and limited studies on Tmem88b itself:

• Mechanistic Insights:

  • Tmem88b is predicted to be involved in negative regulation of canonical Wnt signaling, similar to Tmem88

  • The protein likely functions through interaction with Dishevelled (Dvl) proteins via its C-terminal domain

  • The transmembrane domains may anchor the protein in proximity to Wnt receptors, facilitating regulatory functions

• Experimental Evidence:

  • Studies in zebrafish indicate expression patterns consistent with roles in developmental processes regulated by Wnt signaling

  • Structural predictions suggest conserved domains between Tmem88 and Tmem88b that mediate Wnt pathway interactions

  • Preliminary findings suggest potential tissue-specific regulation of Wnt signaling

• Research Gaps and Future Directions:

  • Direct biochemical evidence of Tmem88b-Dvl interaction is still limited

  • The specific contexts (developmental stage, tissue type) where Tmem88b regulates Wnt signaling require further characterization

  • Potential redundancy or complementarity with Tmem88 needs systematic investigation

To further elucidate this relationship, co-immunoprecipitation studies with Wnt pathway components, TOPFlash reporter assays, and genetic studies using CRISPR-mediated knockouts would provide valuable insights.

What is known about Tmem88b expression in neuronal tissues and its potential role in neuropsychiatric conditions?

Recent research has begun to explore Tmem88b expression in neuronal tissues and its potential implications for neuropsychiatric conditions:

• Expression Patterns:

  • Transcriptomic analyses have detected Tmem88b expression in the medial prefrontal cortex (mPFC) of mice

  • Expression levels appear to be modulated by early life stress (ELS), suggesting potential involvement in stress-responsive neuronal circuits

  • Developmental expression patterns indicate temporal regulation during critical periods of brain development

• Functional Implications:

  • Preliminary research suggests correlation between Tmem88b expression and social behavior phenotypes

  • Altered expression has been observed in conditions affecting neuronal architecture in the mPFC

  • Potential role in Wnt signaling within neuronal contexts may influence synaptic plasticity and neuronal morphology

• Relationship to Neuropsychiatric Conditions:

  • Early life stress models show transcriptomic alterations that include Tmem88b pathway components

  • Changes in social hierarchical behavior correlate with gene expression changes that may involve Tmem88b

  • The protein's potential role in neuronal development through Wnt pathway regulation suggests relevance to neurodevelopmental disorders

This is an emerging field requiring further investigation through conditional knockout studies in specific neuronal populations, electrophysiological assessments, and behavioral phenotyping in animal models with altered Tmem88b expression.

How does Tmem88b compare across species and what can be learned from evolutionary conservation?

Comparative analysis of Tmem88b across species reveals important evolutionary insights:

• Cross-Species Conservation:

  • Tmem88b is conserved across vertebrates, with orthologs identified in humans (UniProt ID: A6NKF7), zebrafish, chicken, and other species

  • The transmembrane domains show the highest degree of conservation, suggesting functional importance

  • The C-terminal domain contains conserved motifs likely involved in protein-protein interactions

• Sequence Homology Analysis:

  Species	Sequence Identity to Mouse Tmem88b	Protein Length	Key Differences
  Human	~85-90%	163 aa	Shorter C-terminus
  Rat	~95-98%	173 aa	Highly conserved
  Zebrafish	~60-65%	221-235 aa	Extended N-terminus
  Chicken	~70-75%	Variable	Species-specific insertions
  Bovine	~80-85%	166 aa	Minor sequence variations

• Functional Conservation:

  • The predicted role in Wnt signaling regulation appears conserved across species

  • Expression patterns show both conserved and species-specific features

  • Human TMEM88B shows 96% sequence identity in certain functional domains compared to mouse

• Evolutionary Insights:

  • The Tmem88/Tmem88b duplication likely occurred early in vertebrate evolution

  • Different selective pressures across species suggest tissue-specific functional specialization

  • Conservation patterns highlight potentially critical functional domains for targeted studies

Evolutionary analysis provides valuable guidance for experimental design, highlighting conserved regions as targets for functional studies and suggesting that findings in mouse models may have translational relevance to human biology.

What are the key considerations for designing reproducible experiments with Tmem88b in mouse models?

When designing mouse experiments involving Tmem88b, follow these guidelines to ensure reproducibility:

• Strain Selection and Genetic Background:

  • Carefully choose mouse strains based on experimental needs and document strain information completely

  • Consider that mouse strains "are as variable as dog breeds" and have distinct characteristics that may impact results

  • Document genetic background thoroughly and maintain consistency across experiments

• Experimental Design Framework:

  • Follow the 3Rs principles (Replacement, Refinement, Reduction) plus the "4th R" - Reproducibility

  • Use tools like the NC3Rs Experimental Design Assistant for planning studies

  • Prepare a detailed study plan following ARRIVE guidelines to document all aspects of the experimental design

• Sample Size and Statistical Power:

  • Perform power calculations to determine appropriate sample sizes before beginning experiments

  • Account for potential attrition and variability between animals

  • Plan for adequate biological replicates (different mice) rather than just technical replicates

• Controls and Variables:

  • Implement proper controls for all experiments (positive, negative, vehicle)

  • Remember that "despite being more-or-less genetically identical within a particular strain, [mice] can show phenotypic variability"

  • Control for environmental factors, including housing conditions, handling procedures, and time of day for experiments

• Documentation and Reporting:

  • Document all procedures in detail, including any deviations from planned protocols

  • Report all results, including negative findings and unexpected observations

  • Include comprehensive methods sections that enable others to replicate your work

Following these guidelines will significantly improve the reproducibility and reliability of Tmem88b research in mouse models.

What protocols should be followed for proper handling and storage of recombinant Tmem88b?

For optimal handling and storage of recombinant Tmem88b, follow these evidence-based protocols:

• Initial Receipt and Processing:

  • Upon receipt, immediately store according to manufacturer recommendations

  • For lyophilized protein, reconstitute in sterile PBS to a concentration of 100 μg/mL (similar to recommendations for other recombinant proteins)

  • Filter through a 0.22 μm filter if necessary to ensure sterility

• Storage Conditions:

  • Short-term storage (up to 1 week): 4°C in PBS buffer

  • Long-term storage: -20°C to -80°C

  • Avoid repeated freeze-thaw cycles; store in small single-use aliquots

  • For lyophilized preparations, maintain at -20°C in a desiccated environment

• Working with the Protein:

  • Thaw aliquots rapidly at room temperature or 37°C water bath

  • Keep on ice when working at the bench

  • Centrifuge briefly before opening tubes to collect all material

  • Use low-binding tubes to minimize protein loss

• Stability Considerations:

  • Product stability is typically maintained for 6 months when stored properly

  • Activity loss should be less than 5% within the expiration date under appropriate storage conditions

  • Consider adding stabilizers like 50% glycerol for freeze-thaw protection

  • BSA addition (0.1-1%) may improve stability for dilute solutions, unless using for applications where BSA would interfere

• Quality Control:

  • Periodically verify protein integrity using SDS-PAGE

  • For functional studies, include activity controls with each experiment

  • Document all handling procedures and storage times

Following these protocols will help maintain the integrity and activity of recombinant Tmem88b for your research applications.

How should I approach troubleshooting expression or functional issues with recombinant Tmem88b?

When troubleshooting recombinant Tmem88b expression or functional issues, follow this systematic approach:

• Expression Problems:

  Problem	Potential Causes	Troubleshooting Approaches
  Low yield	Toxicity to expression host; poor codon optimization; protein degradation	Try different expression systems; optimize codons; add protease inhibitors; lower induction temperature
  Inclusion bodies	Improper folding; overexpression	Reduce expression rate; co-express chaperones; optimize lysis conditions; consider refolding protocols
  Proteolytic degradation	Host proteases; sample handling	Add protease inhibitors; optimize purification speed; check storage conditions
  Poor solubility	Transmembrane nature of protein	Include appropriate detergents; try different solubilization buffers; consider fusion tags that enhance solubility

• Purification Challenges:

  • For affinity tag issues: Verify tag accessibility; adjust imidazole concentrations for His-tag purification

  • For aggregation: Try different detergents; optimize buffer composition; consider on-column folding

  • For co-purifying contaminants: Add washing steps with increased salt or detergent; consider secondary purification methods

  • For low purity: Implement multi-step purification strategy to achieve >80% purity

• Functional Assays Troubleshooting:

  • No activity: Verify protein folding; check buffer compatibility; ensure proper post-translational modifications

  • Variable results: Standardize protein concentration determination; use fresh aliquots; control experimental conditions

  • Interference issues: Use carrier-free preparations for sensitive assays ; control for tag effects

• Systematic Approach:

  • Document all variables and changes systematically

  • Test one parameter at a time

  • Include appropriate positive and negative controls

  • Consider consulting specific guidelines for transmembrane protein work

• Validation Methods:

  • Confirm protein identity via mass spectrometry or N-terminal sequencing

  • Assess structural integrity using circular dichroism or limited proteolysis

  • Verify activity using established functional assays

  • Check endotoxin levels if working with cell-based assays (<1.0 EU per μg)

Remember that as a transmembrane protein, Tmem88b presents specific challenges that may require specialized approaches compared to soluble proteins.

What regulatory and biosafety considerations apply to research with recombinant Tmem88b?

Research with recombinant Tmem88b must comply with various regulatory and biosafety requirements:

• NIH Guidelines Compliance:

  • Research with recombinant Tmem88b falls under the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules

  • Experiments require Institutional Biosafety Committee (IBC) approval before initiation

  • The specific section that applies depends on the nature of your work:

    • Section III-D for standard recombinant protein expression in laboratory strains

    • Section III-C for work involving human subjects

    • Section III-B for experiments involving toxin molecules

• Biosafety Level Determinations:

  • Standard work with recombinant Tmem88b typically falls under Biosafety Level 1 (BL1) containment

  • Risk assessment should consider:

    • Source of the genetic material

    • Host-vector systems used

    • Protein's biological activity

    • Scale of operation

• Institutional Requirements:

  • Submit protocols to your Institutional Biosafety Committee

  • Ensure all personnel have appropriate training

  • Document risk assessments and containment procedures

  • Follow institutional waste management protocols

• International Considerations:

  • For research conducted outside the United States with NIH funding, both local regulations and NIH guidelines must be followed

  • If the host country lacks relevant regulations, NIH-approved safety practices must still be implemented

• Special Precautions:

  • For animal experimentation, follow ARRIVE guidelines for study planning

  • Product documentation clearly states "Not For Human Consumption"

  • For CRISPR applications targeting Tmem88b, additional review may be required

Compliance with these regulations not only ensures legal operation but also promotes safe and responsible research practices.