Laboratory Methods
BIOINFORMATICS
CoSMoS.c.This algorithm was developed by Shuang Li to search for and identify amino acid motifs that are highly conserved across 1002 yeast strains. For further details refer to this paper: Li S, Dohlman HG. Evolutionary conservation of sequence motifs at sites of protein modification. J Biol Chem. 2023 May;299(5):104617. doi: 10.1016/j.jbc.2023.104617. Epub 2023 Mar 16. PMID: 36933807; PMCID: PMC10139944.
pHinder. This algorithm searches protein structures for buried proton-sensing motifs. Please contact Dan Isom for details and refer to this paper: Isom DG, Sridharan V, Baker R, Clement ST, Smalley DM, Dohlman HG. Protons as second messenger regulators of G protein signaling. Mol Cell. 2013 Aug 22;51(4):531-8. doi: 10.1016/j.molcel.2013.07.012. Epub 2013 Aug 15. PMID: 23954348; PMCID: PMC3770139.
QUANTITATION OF THE YEAST MATING RESPONSE
Liquid Beta-galactosidase Reporter Gene Assay : This protocol is used to quantitatively measure the pheromone response over a range of pheromone concentrations.
Pheromone Halo Assay : This is a bio-assay that measures the responsiveness of cells to a factor pheromone. The assay is easy to conduct and the results are usually unambiguous and highly reproducible.
TCA Whole Cell Extracts : This is a quantitative method for extracting total proteins for Western Blotting, especially good with phospho-MAPK antibodies.
SDS Whole Cell Extracts : This is a simple and rapid method for extracting total proteins from Yeast for SDS-PAGE and Western Blotting.
Beta-galactosidase Filter Assay : This method has been used in our lab to screen for and examine mutants with altered pheromone responses.
For further details please refer to this paper: Shellhammer JP, Pomeroy AE, Li Y, Dujmusic L, Elston TC, Hao N, Dohlman HG. Quantitative analysis of the yeast pheromone pathway. Yeast. 2019 Aug;36(8):495-518. doi: 10.1002/yea.3395. Epub 2019 Jun 27. PMID: 31022772; PMCID: PMC6684483.
PURIFICATION AND ISOLATION
GST Fusion Protein Purification from Yeast : This protocol has be used to assay binding of Sst2-RGS and Gbeta/gamma to Gpa1-GST.
TAP Fusion Protein Purification from Yeast
FLAG Fusion Protein Purification from Yeast
Sucrose Density Gradient Fractionation of Yeast Membranes: This is a protocol for localization of membrane associated proteins in yeast. For details refer to this paper: Song J, Hirschman J, Gunn K, Dohlman HG. Regulation of membrane and subunit interactions by N-myristoylation of a G protein alpha subunit in yeast. J Biol Chem. 1996 Aug 23;271(34):20273-83. doi: 10.1074/jbc.271.34.20273. PMID: 8702760.
Preparation of Yeast cells for FACS analysis
MOLECULAR BIOLOGY METHODS
“Lazy Bones” PLATE Transformation : This is a quick method for plasmid transformation into yeast.
Plasmid Isolation from Yeast using Qiagen “QiaPrep” Kit
Plasmid Library Amplification : This protocol has been used successfully in our lab to amplify a LEU2 CEN library. After several screens we determined that the quality of the amplified library was excellent.