Recombinant Rat UPF0767 protein C1orf212 homolog

What is UPF0767 protein C1orf212 homolog and why is it relevant to research?

UPF0767 protein C1orf212 homolog, also known as Smim12 (Small integral membrane protein 12), is a protein of interest in molecular biology research. The "UPF" prefix indicates it belongs to a group of uncharacterized protein families, suggesting its functions are still being elucidated. This protein is relevant to research because characterizing novel proteins advances our understanding of cellular pathways and potential therapeutic targets. The rat homolog has the UniProt accession number D4ACP2, while the mouse homolog is identified as Q78RX3 . Research with recombinant versions of this protein enables functional studies, antibody production, and structural analyses that can reveal its biological roles.

What are the storage and stability characteristics of Recombinant Rat UPF0767 protein C1orf212 homolog?

The stability and shelf life of Recombinant Rat UPF0767 protein C1orf212 homolog depends on several factors including storage conditions, buffer composition, and protein formulation. For optimal preservation:

• Liquid formulations typically maintain stability for approximately 6 months at -20°C/-80°C

• Lyophilized formulations have extended stability up to 12 months at -20°C/-80°C

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

• Repeated freeze-thaw cycles significantly reduce protein integrity and should be avoided

For long-term storage, it is recommended to prepare aliquots with 5-50% glycerol (with 50% being the standard recommendation) and store at -20°C/-80°C to prevent degradation while minimizing freeze-thaw damage .

How should Recombinant Rat UPF0767 protein C1orf212 homolog be reconstituted for experimental use?

Proper reconstitution is essential for maintaining protein activity. The recommended protocol includes:

• Briefly centrifuge the vial prior to opening to collect contents at the bottom

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

• Add glycerol to a final concentration of 5-50% (typical standard is 50%)

• Prepare multiple small aliquots to minimize freeze-thaw cycles

• Store reconstituted aliquots at -20°C/-80°C for long-term storage

This approach maximizes protein stability while minimizing degradation from repeated handling. When working with the protein, maintain sterile conditions and use appropriate buffers to preserve structural integrity and function.

What are the optimal expression systems for producing Recombinant Rat UPF0767 protein C1orf212 homolog?

Different expression systems offer varying advantages for recombinant protein production. Based on available data:

The choice of expression system should be determined by the specific experimental requirements. For structural studies requiring high purity and yield, E. coli systems may be preferred. For functional studies requiring mammalian-like modifications, yeast or mammalian expression systems are more appropriate. The recombinant rat protein is available from commercial sources produced in yeast systems with >85% purity as determined by SDS-PAGE .

What protein purification strategies are most effective for isolating Recombinant Rat UPF0767 protein C1orf212 homolog?

Purification strategies depend on the tagging system and expression host. Based on available commercial products:

• Affinity Chromatography: His-tagged versions can be purified using nickel or cobalt resin columns, which is evident in the mouse homolog product (His-tagged)

• Size Exclusion Chromatography: Often used as a polishing step after initial purification to achieve >85-90% purity

• Buffer Optimization:

  • For storage: Tris/PBS-based buffers with 6% Trehalose at pH 8.0 maintain stability

  • For reconstitution: PBS or deionized water with 5-50% glycerol

• Validation Methods:

  • SDS-PAGE under reducing and non-reducing conditions to confirm protein integrity

  • Functional ELISA to verify binding capacity (as demonstrated with EGFR proteins)

For highest experimental reproducibility, researchers should verify protein purity via SDS-PAGE and may need to perform additional purification steps depending on the specific application requirements.

What are the recommended methods for analyzing UPF0767 protein C1orf212 homolog interactions with potential binding partners?

Several methodological approaches can be employed to study protein-protein interactions:

• Co-Immunoprecipitation (Co-IP):

  • Utilize antibodies against UPF0767 protein C1orf212 homolog to pull down potential interacting proteins

  • Analyze pulled-down complexes via mass spectrometry to identify binding partners

• Surface Plasmon Resonance (SPR):

  • Immobilize purified UPF0767 protein C1orf212 homolog on sensor chips

  • Measure real-time binding kinetics with potential interacting proteins

  • Similar to the ELISA binding studies reported for EGFR proteins which demonstrated binding with ED50 values in the μg/mL range

• Yeast Two-Hybrid Screening:

  • Construct bait plasmids containing UPF0767 protein C1orf212 homolog

  • Screen against cDNA libraries to identify potential interacting proteins

• Functional ELISA:

  • Similar to methods described for EGFR protein binding assays

  • Plate-bound UPF0767 protein can be tested against potential binding partners

When designing interaction studies, it's important to consider the protein's potential membrane association (given its classification as Smim12 - Small integral membrane protein 12) and include appropriate detergents or lipid environments to maintain native conformation.

How can researchers validate antibodies against Recombinant Rat UPF0767 protein C1orf212 homolog?

Antibody validation is critical for ensuring experimental reproducibility. A comprehensive validation strategy includes:

• Western Blot Analysis:

  • Positive control: Recombinant rat UPF0767 protein at known concentrations

  • Negative control: Lysates from tissues/cells known not to express the protein

  • Expected molecular weight should align with the predicted size of the protein

• Immunoprecipitation Efficiency Testing:

  • Spike recombinant protein into complex lysates

  • Determine recovery percentage after immunoprecipitation

  • Verify specificity by mass spectrometry analysis of pulled-down proteins

• Cross-Reactivity Assessment:

  • Test against homologs from other species (mouse, dog variants are available )

  • Determine species specificity for experimental planning

• Peptide Competition Assays:

  • Pre-incubate antibodies with excess peptide antigen

  • Confirm signal reduction in subsequent immunoassays

• Knockout/Knockdown Validation:

  • Compare antibody reactivity in wild-type vs. knockout/knockdown systems

  • Absence of signal in knockout systems confirms specificity

Importantly, researchers should document all validation experiments and include appropriate controls when using antibodies in experimental workflows.

What are the known or predicted functions of UPF0767 protein C1orf212 homolog?

• Its alternative name, Small integral membrane protein 12 (Smim12), suggests membrane localization

• The amino acid sequence (available for the mouse homolog) contains hydrophobic regions consistent with membrane integration

• As a C1orf212 homolog, it may share functions with the human chromosome 1 open reading frame 212 protein

Research approaches to characterize function might include:

• Gene knockout/knockdown studies to observe phenotypic effects

• Proteomics analysis to identify interaction partners

• Structural studies to infer function from protein folding patterns

• Subcellular localization experiments to determine cellular compartments where the protein functions

Given the limited published data on this protein's function, researchers investigating UPF0767 protein C1orf212 homolog have opportunities for novel discoveries regarding its biological role.

How can Recombinant Rat UPF0767 protein C1orf212 homolog be utilized in cellular assays?

The recombinant protein can be employed in various cellular assays to investigate function:

• Cellular Uptake Studies:

  • Fluorescently label the recombinant protein

  • Track internalization patterns using confocal microscopy

  • Compare with other membrane proteins to identify trafficking pathways

• Competitive Binding Assays:

  • Similar to methodology used for chemokine studies

  • Determine if UPF0767 competes with known ligands of membrane receptors

• Membrane Integration Analysis:

  • Use recombinant protein in artificial membrane systems

  • Measure integration efficiency and orientation

  • Determine effects on membrane properties

• Cell Signaling Impact:

  • Treat cells with recombinant protein at various concentrations

  • Measure changes in downstream signaling pathways

  • Similar to approaches used with other recombinant proteins like EGFR

When designing cellular assays, researchers should consider the potential membrane association of the protein and account for this in their experimental conditions, including appropriate detergents and buffer systems.

What are the recommended approaches for studying species-specific differences in UPF0767 protein C1orf212 homologs?

Comparative analysis across species provides valuable insights into evolutionary conservation and functional importance. Recommended approaches include:

• Sequence Alignment Analysis:

  • Compare amino acid sequences from rat, mouse, dog, and other species

  • Identify conserved domains and species-specific variations

  • Available sequences from recombinant protein sources provide a starting point

• Functional Conservation Testing:

  • Express homologs from different species in cellular systems

  • Compare localization patterns and binding partners

  • Assess functional complementation in knockout models

• Structural Comparison:

  • Generate 3D models of homologs from different species

  • Compare predicted structural elements

  • Identify conserved binding pockets or interaction surfaces

• Cross-Species Binding Studies:

  • Test if binding partners of one species' homolog interact with others

  • Quantify binding affinity differences using SPR or ELISA

  • Similar to methodology described for EGFR binding studies

This comparative approach can reveal evolutionarily important domains and species-specific adaptations of the protein, providing clues to its biological function and importance.

What are common challenges when working with recombinant membrane proteins like UPF0767 protein C1orf212 homolog?

Membrane proteins present unique challenges in research settings. For UPF0767 protein C1orf212 homolog (Smim12), researchers might encounter:

• Solubility Issues:

  • Solution: Use appropriate detergents or lipid nanodiscs to maintain protein solubility

  • Consider buffer optimization with trehalose (6%) as used in commercial preparations

• Aggregation During Storage:

  • Solution: Add glycerol (5-50%) to storage buffers as recommended

  • Maintain at recommended temperatures (-20°C/-80°C for long-term)

  • Avoid repeated freeze-thaw cycles

• Loss of Functional Activity:

  • Solution: Validate protein functionality after reconstitution using binding assays

  • Similar to the approach used for EGFR proteins, where binding ability is measured

• Proper Reconstitution:

  • Solution: Follow manufacturer recommendations for centrifugation and buffer composition

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL concentration

• Non-specific Binding in Assays:

  • Solution: Include appropriate blocking agents in assay buffers

  • Use carrier-free preparations for applications where BSA might interfere

Documenting troubleshooting steps and optimized protocols is essential for reproducible research with membrane proteins.

How can researchers optimize Western blot detection of UPF0767 protein C1orf212 homolog?

Western blot optimization for potentially challenging proteins like UPF0767 protein C1orf212 homolog requires attention to several factors:

• Sample Preparation:

  • Include appropriate detergents for membrane protein solubilization

  • Consider using urea-based buffers for complete denaturation

  • Sonicate samples to disrupt potential aggregates

• Gel Selection:

  • For small proteins (mouse homolog is 92 amino acids ), use higher percentage gels (15-20%)

  • Consider gradient gels to resolve potential multimeric forms

• Transfer Conditions:

  • For small membrane proteins, optimize transfer time and voltage

  • Consider using PVDF membranes with smaller pore sizes to prevent protein pass-through

• Blocking Strategy:

  • Test multiple blocking agents (BSA, milk, commercial blockers)

  • Optimize blocking time and temperature

• Antibody Selection and Validation:

  • Verify antibody specificity using recombinant proteins

  • Test multiple antibodies targeting different epitopes

  • Include appropriate positive controls (recombinant protein) in blots

When analyzing results, compare observed molecular weight with expected size. SDS-PAGE analysis of recombinant EGFR proteins showed bands at 110-130 kDa under reducing conditions and 220-260 kDa under non-reducing conditions , suggesting multimerization can occur with some recombinant proteins.

What strategies can address poor expression yields of recombinant UPF0767 protein C1orf212 homolog?

Low expression yields can significantly hamper research progress. Researchers can employ several strategies to improve yields:

• Expression System Optimization:

  • Compare yields between yeast, E. coli, and mammalian systems

  • Commercial sources utilize both yeast and E. coli for different homologs

• Codon Optimization:

  • Adapt codons to match preferred usage in the expression host

  • Particularly important when expressing rat proteins in yeast or E. coli

• Fusion Tag Selection:

  • Test different fusion tags (His, GST, MBP) for improved solubility

  • Commercial products use His-tags for some variants

• Culture Condition Optimization:

  • Adjust temperature, induction timing, and media composition

  • For E. coli, lower temperatures (16-25°C) often improve membrane protein yields

• Truncation Approaches:

  • Express soluble domains if full-length protein yields are consistently low

  • Some commercial products offer partial protein variants

Systematic optimization of these parameters using design of experiments (DOE) approaches can significantly improve recombinant protein yields while maintaining functional integrity.

How can structural biology techniques be applied to characterize UPF0767 protein C1orf212 homolog?

Structural characterization of membrane proteins presents unique challenges but offers valuable insights into function. For UPF0767 protein C1orf212 homolog, potential approaches include:

• X-ray Crystallography:

  • Requires highly pure, homogeneous protein preparations (>90% purity as achieved in commercial products )

  • May require crystallization chaperones or antibody fragments to stabilize structure

  • Detergent selection is critical for membrane protein crystallization

• Cryo-electron Microscopy (Cryo-EM):

  • Increasingly valuable for membrane proteins

  • May reveal structural details without crystallization

  • Can capture multiple conformational states

• Nuclear Magnetic Resonance (NMR):

  • Suitable for smaller proteins (the mouse homolog at 92 amino acids is within range)

  • Requires isotopic labeling (15N, 13C) during recombinant expression

  • Can provide dynamic information about protein movements

• Molecular Dynamics Simulations:

  • Complement experimental approaches

  • Utilize the amino acid sequence available for homologs

  • Predict membrane interactions and conformational flexibility

The choice of method depends on research goals, available resources, and protein properties. Structural information could provide crucial insights into this uncharacterized protein's function.

What are the considerations for developing cell-based assays to study UPF0767 protein C1orf212 homolog?

Cell-based assays provide crucial functional insights but require careful design. Key considerations include:

• Cell Line Selection:

  • Choose lines expressing endogenous binding partners

  • Consider species compatibility (rat vs. mouse vs. human cell lines)

  • Include both expressing and non-expressing lines as controls

• Expression Strategies:

  • Develop stable cell lines with inducible expression

  • Use fluorescent protein tags to track localization

  • Ensure expression levels are physiologically relevant

• Functional Readouts:

  • Identify appropriate cellular responses to measure

  • Consider calcium flux, mitogen-activated protein kinase (MAPK) activation, or other signaling pathways

  • Similar to approaches used for chemokine functional studies

• Knockdown/Knockout Controls:

  • Generate CRISPR/Cas9 knockout cell lines

  • Use siRNA for transient knockdown experiments

  • Compare phenotypes with exogenous protein addition

• High-Content Imaging:

  • Track protein trafficking and localization

  • Measure co-localization with organelle markers

  • Quantify membrane integration efficiency

These considerations ensure that cell-based assays provide meaningful insights into protein function while controlling for experimental variables.