Recombinant Rat Uncharacterized protein C4orf3 homolog

What is Rat Uncharacterized protein C4orf3 homolog?

Rat Uncharacterized protein C4orf3 homolog is a small protein (65 amino acids) with UniProt ID Q498U0. It is the rat ortholog of the human C4orf3 protein (chromosome 4 open reading frame 3), whose function remains largely unknown. The protein is typically expressed in E. coli systems for research purposes, often with an N-terminal His tag to facilitate purification and detection. The designation "uncharacterized" indicates that its biological function has not yet been fully elucidated, making it a potential target for functional genomics studies .

What are the optimal storage and handling conditions?

For optimal stability, Recombinant Rat Uncharacterized protein C4orf3 homolog should be stored at -20°C or -80°C upon receipt. The protein is typically provided as a lyophilized powder and requires proper handling to maintain its integrity. Key storage recommendations include:

• Store the lyophilized protein at -20°C/-80°C

• After reconstitution, prepare working aliquots to avoid repeated freeze-thaw cycles

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

• For long-term storage, add glycerol (5-50% final concentration, with 50% being the standard recommendation)

• The storage buffer typically consists of Tris/PBS-based buffer with 6% Trehalose at pH 8.0, or Tris-based buffer with 50% glycerol

Repeated freeze-thaw cycles should be avoided as they can compromise protein stability and biological activity. Proper aliquoting upon initial reconstitution is therefore essential for maintaining protein quality throughout a research project.

What is the recommended reconstitution protocol?

The reconstitution process is critical for maintaining protein activity. Follow these methodological steps:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

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

• For long-term storage, add glycerol to a final concentration of 5-50% (50% is standard)

• Gently mix until completely dissolved

• Prepare small working aliquots to prevent repeated freeze-thaw cycles

• Store reconstituted protein according to the temperature requirements (-20°C/-80°C for long-term storage, 4°C for short-term use)

This methodical approach ensures maximum retention of protein stability and activity for downstream applications such as binding studies, functional assays, or structural analyses.

How can researchers validate protein identity and purity?

Validation of protein identity and purity is essential before proceeding with experiments. Recommended validation methods include:

• SDS-PAGE analysis to confirm molecular weight (approximately 7.1 kDa for the core protein, with additional weight from the His-tag)

• Western blotting using anti-His antibodies or specific antibodies against the protein

• Mass spectrometry for precise molecular weight determination and sequence verification

• Circular dichroism (CD) spectroscopy to assess secondary structure elements

• Size exclusion chromatography to evaluate protein homogeneity

According to manufacturer specifications, the protein purity should be greater than 90% as determined by SDS-PAGE . When designing validation experiments, researchers should consider both tag-based detection methods and techniques that verify the protein's primary sequence.

What bioinformatic approaches can help predict the function of this uncharacterized protein?

Since C4orf3 homolog is uncharacterized, bioinformatic analyses represent crucial first steps in functional prediction. A comprehensive approach would include:

• Sequence homology analysis using BLAST against characterized proteins

• Domain prediction using tools such as PFAM, SMART, or InterPro

• Secondary structure prediction using PSIPRED or JPred

• Tertiary structure modeling using AlphaFold2 or I-TASSER

• Subcellular localization prediction with tools like TargetP, PSORT, or DeepLoc

• Analysis of conserved motifs across species to identify potential functional regions

• Gene ontology (GO) term inference based on structural features

• Protein-protein interaction prediction using tools like STRING

The amino acid sequence (MEVGQAASGTDGVRERRGSSAARRRSQDEPVQSGMNGIPKHSYWLDLWLFILFDLALFIFVYLLP) suggests the presence of a hydrophobic C-terminal region that might indicate membrane association, which could guide experimental designs to investigate its cellular localization and function .

How can researchers design experiments to characterize protein-protein interactions?

To identify potential interaction partners of Rat Uncharacterized protein C4orf3 homolog, consider these methodological approaches:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Utilize the His-tag for pulldown experiments from rat tissue or cell lysates

  • Analyze co-precipitated proteins by mass spectrometry

  • Validate interactions with reciprocal co-immunoprecipitation

• Yeast two-hybrid (Y2H) screening:

  • Generate bait constructs using the full-length protein or domains

  • Screen against rat cDNA libraries

  • Validate positive interactions using alternative methods

• Proximity-dependent biotin identification (BioID) or APEX2:

  • Express the protein fused to BioID2 or APEX2 in relevant cell types

  • Identify neighboring proteins through biotinylation and streptavidin pulldown

• Cross-linking mass spectrometry (XL-MS):

  • Use chemical cross-linkers to capture transient interactions

  • Identify crosslinked peptides through specialized MS protocols

The His-tag present in the recombinant protein provides a convenient handle for many of these interaction studies, particularly for affinity purification approaches . When designing these experiments, researchers should consider both cytosolic and membrane-associated protein extraction protocols, given the potential membrane association indicated by the protein sequence.

What are the predicted structural characteristics based on sequence analysis?

Based on the amino acid sequence, several structural characteristics can be predicted:

These structural predictions can guide experimental design, particularly for structural biology approaches such as X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy . The potential membrane association suggested by the hydrophobic C-terminus would necessitate special considerations for structural studies, including the potential use of detergents or lipid nanodiscs.

What challenges might researchers encounter with protein solubility and stability?

The sequence characteristics of Rat Uncharacterized protein C4orf3 homolog suggest potential challenges in handling:

• Solubility issues: The hydrophobic C-terminal region (WLFILFDLALFIFVYLLP) indicates potential membrane association, which might lead to solubility problems in aqueous buffers. To address this:

  • Consider including mild detergents (0.1% Triton X-100, 0.5% CHAPS) in working buffers

  • Test different buffer compositions with varying salt concentrations (150-500 mM NaCl)

  • Evaluate protein behavior at different pH values (pH 6.5-8.5)

• Stability concerns: Small proteins can be prone to degradation. Strategies to enhance stability include:

  • Add protease inhibitors to all working solutions

  • Maintain cold temperatures during handling (4°C)

  • Include stabilizing agents like glycerol (10-20%) or low concentrations of reducing agents

  • Store at appropriate temperatures (-20°C/-80°C) with 50% glycerol for long-term storage

Methodologically, researchers should perform pilot experiments to determine optimal conditions for their specific application, monitoring protein stability through SDS-PAGE analysis at different time points under various storage conditions.

How can researchers overcome expression and purification challenges?

When working with recombinant C4orf3 homolog, researchers might encounter several expression and purification challenges:

• Expression optimization:

  • Test multiple expression systems (bacterial, insect, mammalian) if native folding is a concern

  • Evaluate different induction conditions (temperature, inducer concentration, duration)

  • Consider codon optimization for the expression host

  • Test fusion partners that might enhance solubility (MBP, SUMO, GST)

• Purification strategies:

  • Immobilized metal affinity chromatography (IMAC) using the His-tag

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography as a polishing step

  • Consider on-column refolding if the protein forms inclusion bodies

• Quality control:

  • Confirm purity by SDS-PAGE (>90% purity expected)

  • Verify identity by mass spectrometry

  • Assess homogeneity by dynamic light scattering

  • Validate tag accessibility by anti-His western blotting

For researchers ordering commercial recombinant protein, quality assessment upon receipt is still recommended to confirm that specifications match the manufacturer's claims.

What approaches are recommended for determining cellular localization?

Determining the cellular localization of Rat Uncharacterized protein C4orf3 homolog is a crucial step in functional characterization. Recommended methodological approaches include:

• Immunofluorescence microscopy:

  • Generate specific antibodies against the protein or use anti-His antibodies

  • Perform co-localization studies with organelle-specific markers

  • Analyze fixed cells from relevant rat tissues or cell lines

• Subcellular fractionation:

  • Isolate different cellular compartments (cytosol, membrane, nucleus, etc.)

  • Detect protein distribution by western blotting

  • Compare observed distribution with bioinformatic predictions

• Fluorescent protein fusion approaches:

  • Generate C- and N-terminal GFP (or variants) fusion constructs

  • Express in relevant cell types and observe live cell localization

  • Compare with predicted localization based on sequence analysis

• Proximity labeling methods:

  • Create fusion proteins with BioID or APEX2

  • Identify neighboring proteins through biotinylation

  • Infer localization based on known localization of interaction partners

The hydrophobic C-terminal region in the protein sequence suggests possible membrane association, which should be specifically investigated using membrane protein extraction protocols and membrane-specific markers .

How should researchers design experiments to identify physiological functions?

To determine the physiological function of Rat Uncharacterized protein C4orf3 homolog, a systematic approach is recommended:

• Loss-of-function studies:

  • Design siRNA or CRISPR-Cas9 strategies targeting the gene

  • Evaluate phenotypic changes in relevant cell types or animal models

  • Perform transcriptomic and proteomic analyses to identify affected pathways

• Gain-of-function studies:

  • Overexpress the protein in relevant cell types

  • Assess changes in cellular phenotypes (morphology, growth, differentiation)

  • Investigate effects on specific signaling pathways based on bioinformatic predictions

• Expression pattern analysis:

  • Examine tissue-specific expression patterns by qRT-PCR or western blotting

  • Correlate expression with specific physiological states or developmental stages

  • Compare with expression patterns of known functional homologs in other species

• Evolutionary conservation analysis:

  • Compare sequence conservation across species

  • Identify highly conserved residues that might be functionally important

  • Design point mutations to test the functional significance of conserved residues

Starting with comprehensive bioinformatic analyses to generate hypotheses about potential functions based on sequence features, domain predictions, and evolutionary conservation would provide direction for targeted experimental approaches.

What structural biology approaches are suitable for this protein?

Determining the three-dimensional structure of Rat Uncharacterized protein C4orf3 homolog could provide valuable insights into its function. Recommended structural biology approaches include:

• X-ray crystallography:

  • Optimize protein expression and purification for high purity and homogeneity

  • Screen crystallization conditions systematically

  • Consider tag removal for crystallization attempts

  • If membrane-associated, use lipidic cubic phase crystallization

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Particularly suitable for small proteins (<20 kDa)

  • Requires 15N/13C-labeled protein production

  • Can provide dynamics information in addition to structure

  • May provide insights into potential ligand binding sites

• Cryo-electron microscopy (cryo-EM):

  • More suitable if the protein forms larger complexes

  • May require fusion to a larger protein scaffold for single-particle analysis

  • Consider using Fab fragments to increase particle size

• Small-angle X-ray scattering (SAXS):

  • Provides low-resolution structural information in solution

  • Can confirm folding and oligomeric state

  • Useful for studying conformational changes

Each approach has advantages and limitations, and the choice depends on specific research questions and available resources. Given the small size of this protein (65 amino acids), NMR spectroscopy might be particularly well-suited for structural characterization .

How can researchers integrate multi-omics approaches to understand protein function?

A comprehensive multi-omics strategy can accelerate functional characterization of Rat Uncharacterized protein C4orf3 homolog:

• Transcriptomics integration:

  • Analyze co-expression networks to identify genes with similar expression patterns

  • Perform differential expression analysis following protein knockdown/overexpression

  • Identify transcription factors that might regulate the gene

• Proteomics approaches:

  • Conduct interaction proteomics using affinity purification-mass spectrometry

  • Analyze post-translational modifications

  • Perform global proteome analysis after gene modulation

• Metabolomics integration:

  • Investigate metabolic changes associated with protein modulation

  • Identify potential metabolic pathways affected

• Integrated data analysis framework:

  • Apply machine learning approaches to integrate multi-omics data

  • Use pathway enrichment analysis across datasets

  • Develop predictive models for protein function based on integrated data

This integrated approach can provide a systems-level understanding of protein function and place it within the broader context of cellular physiology.