Fungi

Serial Immunoprecipitation of 3xFLAG/V5-tagged Yeast Proteins to Identify Specific Interactions with Chaperone Proteins

Authors: Xu Zheng
Xu ZhengAffiliation: Whitehead Institute for Biomedical Research, Cambridge, USA
Bio-protocol author page: a4703
and David Pincus
David PincusAffiliation: Whitehead Institute for Biomedical Research, Cambridge, USA
For correspondence: pincus@wi.mit.edu
Bio-protocol author page: a4704
, date: 6/20/2017, 234 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2348.

[Abstract] This method was generated to isolate high affinity protein complexes from yeast lysate by performing serial affinity purification of doubly tagged 3xFLAG/V5 proteins. First, the bait protein of interest is immunoprecipitated by anti-FLAG-conjugated magnetic beads and gently eluted by 3xFLAG antigen ...

In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease

Authors: Sakshi Chauhan
Sakshi ChauhanAffiliation: Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India
Bio-protocol author page: a3946
, Gajendra Kumar Azad
Gajendra Kumar AzadAffiliation 1: Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India
Affiliation 2: Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel
Bio-protocol author page: a3947
and Raghuvir Singh Tomar
Raghuvir Singh TomarAffiliation: Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India
For correspondence: rst@iiserb.ac.in
Bio-protocol author page: a3948
, date: 1/5/2017, 1265 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2085.

[Abstract] Histone proteins are subjected to a wide array of reversible and irreversible post-translational modifications (PTMs) (Bannister and Kouzarides, 2011; Azad and Tomar, 2014). The PTMs on histones are known to regulate chromatin structure and function. Histones are irreversibly modified by proteolytic ...

Enzymatic Activity Assays in Yeast Cell Extracts

Authors: Melike Çağlayan
Melike ÇağlayanAffiliation: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, USA
Bio-protocol author page: a1849
and Samuel H. Wilson
Samuel H. WilsonAffiliation: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, USA
For correspondence: wilson5@niehs.nih.gov
Bio-protocol author page: a1850
, date: 12/5/2014, 3769 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1312.

[Abstract] Saccharomyces cerevisiae (S. cerevisiae) (commonly known as baker’s yeast) is a model organism that has a similar upstream base excision repair (BER) pathway for the repair of methylated bases as that in mammalian cells, and it is very easy to maintain in the laboratory environment. Here, we described ...

Signaling Assays for Detection of Human G-protein-coupled Receptors in Yeast

Authors: Yasuyuki Nakamura
Yasuyuki NakamuraAffiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan
Bio-protocol author page: a1572
, Jun Ishii
Jun Ishii Affiliation: Organization of Advanced Science and Technology, Kobe University, Kobe, Japan
Bio-protocol author page: a1573
and Akihiko Kondo
Akihiko KondoAffiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan
For correspondence: akondo@kobe-u.ac.jp
Bio-protocol author page: a1574
, date: 8/20/2014, 3489 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1206.

[Abstract] G-protein-coupled receptors (GPCRs) are the largest group of cell-surface proteins and are major molecular targets for drug development. The protocol described herein is for the detection of human GPCR signaling in the yeast Saccharomyces cerevisiae. Using Zoanthus sp. green fluorescent protein (ZsGreen) ...

Ribosome Fractionation in Yeast

Author: Olesya O. Panasenko
Olesya O. PanasenkoAffiliation: Department of Microbiology and Molecular Medicine, University of Geneva, Faculty of Medicine, CMU, Geneva, Switzerland
For correspondence: olesya.panasenko@unige.ch
Bio-protocol author page: a88
, date: 8/20/2012, 9641 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.251.

[Abstract] This protocol describes yeast ribosome fractionation in the gradient of sucrose. During the cyclic process of translation, a small (40S) and large (60S) ribosomal subunit associate with mRNA to form an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation, and then ...

Co-immunoprecipitation in Yeast

Author: Olesya O. Panasenko
Olesya O. PanasenkoAffiliation: Department of Microbiology and Molecular Medicine, University of Geneva, Faculty of Medicine, CMU, Geneva, Switzerland
For correspondence: olesya.panasenko@unige.ch
Bio-protocol author page: a88
, date: 8/20/2012, 10009 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.250.

[Abstract] This protocol describes investigation of protein-protein interactions in baker yeast by co-immunoprecipitation (CoIP). CoIP is a technique to identify physiologically relevant protein-protein interactions in the cell. The interesting protein can be isolated out of solution using antibody that specifically ...

Immunofluorescence Analysis of Yeast Protein

Author: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
, date: 7/5/2012, 7658 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.211.

[Abstract] Many important regulatory proteins such as transcription factors are regulated through subcellular localization. Protein localization can be examined by fusing a GFP tag. However, GFP is relatively big in size, and potentially may affect correct protein localization. Several small tags have been developed, ...

An Improved Method for PAGE-based Detection of Phosphorylated Protein in Yeast

Author: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
, date: 6/20/2012, 7223 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.210.

[Abstract] One dimensional polyacrylamide gel electrophoresis has been successfully used to detect protein phosphorylation. This method is very simple and highly reproducible. Hyperhosphorylated proteins usually migrate slowlier than dephosphorylated proteins. However, not all phosphorylated proteins can be readily ...

In vitro Protein Kinase Assay

Author: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
, date: 6/5/2012, 22685 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.212.

[Abstract] This protocol will describe experimental procedures for an in vitro kinase assay of the yeast protein kinase Sch9. This protocol can be tailored to detect kinase activity of other yeast protein kinase....

Probing Yeast Protein Microarrays for Protein-protein Interactions Using V5-epitope Tagged Fusion Protein Probes

Authors: Joseph Fasolo
Joseph FasoloAffiliation: Department of Genetics, Stanford University, Stanford, USA
Bio-protocol author page: a22
and Michael Snyder
Michael SnyderAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: mpsnyder@stanford.edu
Bio-protocol author page: a1789
, date: 3/5/2012, 6501 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.123.

[Abstract] Protein microarray is probably the only technique currently available for systematic investigation of protein-protein interactions. This protocol describes an optimized method to probe yeast protein microarrays for protein-protein interactions using purified V5-epitope tagged fusion protein. It should ...

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In vitro Protein Kinase Assay

Author: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
, date: 6/5/2012, 22685 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.212.

[Bio101] Protocol for Whole Cell Lysis of Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
, date: 1/5/2011, 19826 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.14.

[Abstract] This protocol describes how to perform lysis on whole yeast cell samples using NaOH. The lysed cells can then be used for downstream applications such as the extraction of total proteins. ...

[Bio101] Large Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicin, Baltimore , USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
, date: 1/5/2011, 11524 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.12.

[Abstract] This protocol can be used to purify membrane proteins from yeast samples under native conditions at a large scale. This protocol has been developed primarily for FLAG-tagged proteins. This protocol can however be slightly modified and applied to other tags, such as GST or HA....

[Bio101] Small Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
, date: 1/5/2011, 11049 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.15.

[Abstract] In this protocol, we show how to purify membrane proteins from yeast using affinity purification under native conditions at a small scale. ...

Co-immunoprecipitation in Yeast

Author: Olesya O. Panasenko
Olesya O. PanasenkoAffiliation: Department of Microbiology and Molecular Medicine, University of Geneva, Faculty of Medicine, CMU, Geneva, Switzerland
For correspondence: olesya.panasenko@unige.ch
Bio-protocol author page: a88
, date: 8/20/2012, 10009 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.250.

Ribosome Fractionation in Yeast

Author: Olesya O. Panasenko
Olesya O. PanasenkoAffiliation: Department of Microbiology and Molecular Medicine, University of Geneva, Faculty of Medicine, CMU, Geneva, Switzerland
For correspondence: olesya.panasenko@unige.ch
Bio-protocol author page: a88
, date: 8/20/2012, 9641 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.251.

[Bio101] Affinity Purification of Yeast Protein-interacting Metabolites for ESI-MS Analysis

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
, date: 4/20/2011, 9460 views, 5 Q&A
DOI: https://doi.org/10.21769/BioProtoc.61.

[Abstract] The method described here can be used to discover in vivo protein-metabolite interactions. Metabolite-protein complexes are purified from yeast cell lysates by an affinity tag that recognizes the protein of interest. The protein-bound metabolites are extracted for identification by mass spectrometry, ...

[Bio101] TAP Purification of Yeast Proteins

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
, date: 1/5/2011, 8891 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.17.

[Abstract] Tandem affinity purification (TAP) is used to look at protein-protein interaction. Its use relies on generating a fusion protein with a TAP tag on the C- or N- terminal end. In this protocol, a two-step purification of N-terminus TAP-tagged proteins from yeast is described....

[Bio101] GST-tagged Yeast Protein Purification

Author: Hogune Im, date: 10/5/2011, 8884 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.141.

[Abstract] Glutation S-transferase (GST) tagging is the most commonly used purification strategy for recombinant protein. It was developed with the goal of preserving the enzymatic activity by utilizing gentle elution condition of the target protein from purification matrix (Poon and Hunt., 1994). The method ...

[Bio101] In-Solution Digestion Of Purified Yeast Protein For LC-MS

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford , USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
, date: 4/20/2011, 8339 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.62.

[Abstract] This method describes the preparation of total yeast protein extract for mass spectrometry analysis. The protein extract is digested by trypsin in a solution with strong denaturants. The digested sample is dried and re-constituted in a mixture compatible with HPLC separation. Samples of isobaric labels ...

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Fungi

Saccharomyces cerevisiae

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Author: Xiyan Li Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USAFor correspondence: lixiyan@stanford.eduBio-protocol author page: a13, date: 4/20/2011, 9460 views, 5 Q&ADOI: https://doi.org/10.21769/BioProtoc.61.

Author: Hogune Im, date: 10/5/2011, 8884 views, 2 Q&ADOI: https://doi.org/10.21769/BioProtoc.141.

Author: Xiyan Li Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford , USAFor correspondence: lixiyan@stanford.eduBio-protocol author page: a13, date: 4/20/2011, 8339 views, 0 Q&ADOI: https://doi.org/10.21769/BioProtoc.62.

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
, date: 4/20/2011, 9460 views, 5 Q&A
DOI: https://doi.org/10.21769/BioProtoc.61.

Author: Hogune Im, date: 10/5/2011, 8884 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.141.

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford , USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
, date: 4/20/2011, 8339 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.62.