Uptake Assay for Radiolabeled Peptides in Yeast
酵母菌中放射性标记肽的吸收测定
发布: 2016年11月20日第6卷第22期 DOI: 10.21769/BioProtoc.2026 浏览次数: 9103
评审: Yanjie LiChong HeMelike Çağlayan
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参见作者原研究论文
The authors used this protocol in:
Oct 2007
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Abstract
We describe an assay for measuring the uptake of radioactive peptides into the yeast Saccharomyces cerevisiae. The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems.
Background
Di/tripeptides and larger oligopeptides are sources of amino acids for protein synthesis, or may serve as carbon and nitrogen precursors for energy production and biosynthesis of metabolites in all organisms. Uptake of di/tripeptides and oligopeptides across the cell membrane is facilitated by peptide transporters. Measuring the accumulation of radiolabeled peptide provides an experimental approach to determine uptake across a specific peptide transport protein in a variety of cell types, including the yeast Saccharomyces cerevisiae. Appropriate experimental design allows for the determination of kinetic parameters such as the affinity and capacity of system under investigation. Dipeptides, such as radiolabeled Leu-Leu, can be used to measure transport across the di/tripeptide transporter Ptr2p of S. cerevisiae. We use the transport of dipeptides across Ptr2p in yeast as an example.
Materials and Reagents
- Culture tube, up to 17 x 100 mm (Thermo Fisher Scientific, Fisher Scientific, catalog number: 14-956-6B )
- 1.5 ml microfuge tubes
- HAWP membrane filter (EMD Millipore, catalog number: HAWP02400 )
- Saccharomyces cerevisiae (S. cerevisiae): such as W303 strain (MATa or MATα leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15) (ATCC, catalog number: 208352 )
Note: Other strains can be used, although the growth medium will have to be adjusted if specific auxotrophic requirements are needed. - Proline (Sigma-Aldrich, catalog number: P5607 )
- Yeast nitrogen base without (NH4)2SO4 and amino acids (BD, catalog number: 233510 )
- D-glucose (Sigma-Aldrich, catalog number: G7021 )
- Uracil (Sigma-Aldrich, catalog number: U1128 )
- Adenine hemisulfate salt (Sigma-Aldrich, catalog number: A9126 )
- Histidine-HCl (Sigma-Aldrich, catalog number: H-8000 )
- Leucine (Sigma-Aldrich, catalog number: L8000 )
- Tryptophan (Sigma-Aldrich, catalog number: T0254 )
- Radiolabeled dipeptide [3H]Leu-Leu (Custom synthesized)
- Leu-Leu (Sigma-Aldrich, catalog number: L-2752 )
- Minimal Proline (MP) broth (see Recipes)
- MP+ broth (see Recipes)
- 20% glucose (see Recipes)
- 2% glucose (see Recipes)
- 2x uptake medium (see Recipes)
Equipment
- 250 ml flask
- Vortex mixer (VWR, catalog number: 97043-562 )
- Water bath (VWR, catalog number: 89501-476 )
- Microscope
- Incubator (set up at 30 °C) containing a rotator mixer (Thermo Fisher ScientificTM, Thermo Scientific model: 1640Q )
- Shaker incubator, 30 °C (Eppendorf, New Brunswick Scientific, model: C-25 )
- Hemocytometer
- Dry bath, 30 °C with blocks to hold microfuge 1.5 ml tubes (Thermo Fisher Scientific, catalog number: 88870002 )
- Manifold vacuum filtration cell harvester (EMD Millipore, catalog number: xx2702550 )
- Filter forceps (EMD Millipore, catalog number: XX6200006P )
- Liquid Scintillation vial, 6 ml Omni-vial with cap (Wheaton, catalog number: 225414 )
- Liquid Scintillation Cocktail (MP Biomedical CytoScint, catalog number: 882453 )
- Liquid Scintillation Counter, TRI-CARB 2000TR (PerkinElmer, catalog number: B291000 )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Hauser, M., Cai, H., Naider, F. and Becker, J. M. (2016). Uptake Assay for Radiolabeled Peptides in Yeast. Bio-protocol 6(22): e2026. DOI: 10.21769/BioProtoc.2026.
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分类
微生物学 > 微生物细胞生物学 > 基于细胞的分析方法
细胞生物学 > 基于细胞的分析方法 > 转运
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