Recombinant Xenopus tropicalis Secretory carrier-associated membrane protein 5 (scamp5)
Description
Functional Roles and Research Findings
SCAMP5 regulates membrane dynamics and interacts with calcium channels and autophagy pathways. Studies highlight its dual role in:
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Calcium channel modulation: SCAMP5 suppresses T-type calcium currents (Ca<sub>v</sub>3.1, Ca<sub>v</sub>3.2, Ca<sub>v</sub>3.3) by reducing channel expression in the plasma membrane .
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Autophagy-exosome coordination: SCAMP5 inhibits autophagosome-lysosome fusion while promoting exosome-mediated protein secretion, a mechanism critical under cellular stress .
Table 2: Key functional interactions of SCAMP5
Production and Purification
Recombinant Xenopus tropicalis SCAMP5 is produced using diverse expression systems:
Table 3: Production platforms for recombinant SCAMP5
Research Applications
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Neurological disease models: SCAMP5 missense mutations (e.g., R91W, G180W) are linked to neurodevelopmental disorders and Parkinson’s disease . Recombinant Xenopus SCAMP5 enables mechanistic studies of these mutations.
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Autophagy-exosome crosstalk: Used to explore how cells switch between protein degradation pathways under stress .
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Evolutionary studies: Comparative analysis with mammalian SCAMP5 homologs reveals conserved secretory pathway mechanisms .
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them during order placement, and we will accommodate your request.
Note: All proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, liquid form has a shelf life of 6 months at -20°C/-80°C. Lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: xtr:493330
UniGene: Str.2318
Q&A
What is the biological role of SCAMP5 in Xenopus tropicalis cellular processes?
SCAMP5 facilitates vesicular trafficking between the trans-Golgi network and plasma membrane, mediating cargo sorting and membrane fusion events. Its tetraspanin structure (four transmembrane domains) enables interactions with SNARE proteins and calcium-sensitive regulators . Researchers confirm its role via:
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Knockdown assays: Morpholino oligonucleotides targeting scamp5 mRNA disrupt secretory vesicle formation in oocytes .
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Co-immunoprecipitation: SCAMP5 binds Syntaxin-1A (STX1A) in Xenopus egg extracts, validated by Western blotting .
Table 1: Recombinant SCAMP5 Protein Specifications
Why is Xenopus tropicalis preferred over X. laevis for SCAMP5 studies?
X. tropicalis offers a diploid genome (2n = 20), simplifying genetic manipulation compared to the pseudotetraploid X. laevis (4n = 36) . Key advantages:
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Transgenic efficiency: 80–90% germline transmission rates using I-SceI meganuclease .
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Gene homology: 85% amino acid identity between X. tropicalis and mammalian SCAMP5 .
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Developmental synchrony: Embryos develop uniformly, enabling high-throughput screens for secretory defects .
How to optimize recombinant SCAMP5 expression in E. coli?
Maximize soluble protein yield using:
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Codon optimization: Replace rare Xenopus tRNA codons (e.g., AGG/AGA arginine) with E. coli-preferred equivalents .
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Induction conditions: Low-temperature induction (16°C) with 0.5 mM IPTG reduces inclusion body formation .
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Buffer additives: 6% trehalose in storage buffers prevents aggregation during lyophilization .
How to resolve discrepancies between predicted and observed SCAMP5 molecular weights?
Discrepancies often arise from post-translational modifications (PTMs) or alternative splicing:
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PTM analysis: Treat purified SCAMP5 with PNGase F to remove N-linked glycosylation; observe ~2 kDa shift via SDS-PAGE .
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Isoform sequencing: Compare cDNA sequences from X. tropicalis oocytes (e.g., ENSXETT00000023355 vs. ENSXETT00000075981) to identify splice variants .
Table 2: SCAMP5 Isoforms in X. tropicalis
| Isoform | Length (aa) | Unique Domains | Expression Pattern |
|---|---|---|---|
| ENSXETP...355 | 231 | Full-length tetraspanin | Oocytes, neural tissue |
| ENSXETP...981 | 189 | Truncated C-terminal domain | Early embryos |
What controls are essential for SCAMP5 localization studies in live cells?
To avoid artifacts in fluorescence-based tracking:
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Endogenous tag validation: Compare CRISPR knock-in mCherry-SCAMP5 lines with overexpression constructs .
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Compartmental markers: Co-stain with Golgi (GM130) and early endosome (EEA1) antibodies .
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FRAP controls: Verify fluorescence recovery after photobleaching in the Golgi apparatus (<60% recovery indicates stable membrane association) .
How to address functional redundancy between SCAMP5 and SCAMP2/3 in secretion assays?
Redundancy complicates phenotype interpretation in knockout models. Solutions include:
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Double/Triple Knockouts: Use CRISPR-Cas9 to delete scamp2, scamp3, and scamp5 in F0 mosaics .
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Dominant-negative mutants: Overexpress SCAMP5 lacking the N-terminal cytoplasmic domain (Δ1–45 aa) to disrupt oligomerization .
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Transcriptomic profiling: RNA-seq of SCAMP5-depleted cells identifies compensatory upregulation of scamp1/4 .
What experimental strategies validate SCAMP5 antibody specificity?
Commercial antibodies often cross-react with SCAMP paralogs. Rigorous validation requires:
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Knockout validation: Western blotting using scamp5 CRISPR-Cas9 KO lysates .
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Peptide competition: Pre-incubate antibody with 10x molar excess of immunizing peptide (e.g., residues 150–165) .
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Immunogold EM: Confirm subcellular localization in Xenopus oocytes .
How to troubleshoot low SCAMP5 solubility in in vitro assays?
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Detergent screening: Test n-dodecyl-β-D-maltopyranoside (DDM) vs. lauryl maltose neopentyl glycol (LMNG) at 1–2× CMC .
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Redox optimization: Include 2 mM TCEP to stabilize cysteine-rich regions .
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Lipid supplementation: Reconstitute SCAMP5 into liposomes with 20% cholesterol to mimic native membranes .
What bioinformatics tools analyze SCAMP5 evolutionary conservation?
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Phylogenetic trees: Use MEGA-X to align SCAMP5 sequences across 12 vertebrates .
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ConSurf: Identify conserved residues in the second luminal loop (critical for SNARE binding) .
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3D homology modeling: SWISS-MODEL templates based on rat SCAMP1 (PDB: 6V7X) .
How to design a SCAMP5 structure-function study using mutagenesis?
Focus on domains implicated in trafficking:
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Site-directed mutagenesis: Cysteine-to-alanine substitutions in the Cys-rich motif (C158A/C162A) disrupt disulfide bonding .
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Circular dichroism: Compare wild-type and mutant SCAMP5 spectra to assess secondary structure loss .
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Functional rescue: Inject mutant mRNA into scamp5 MO embryos; quantify secretory vesicle rescue via electron microscopy .
