재료 및 방법 <ol><li> 효모 종자 <ul><li> 이 연구에 사용된 모든 종자는 BY4742 배경에 근거하고 있습니다. SILAC 제어 스트레인 (매트, his3, leu2, ura3, lys2 및 arg4 : G418)는 : BY4742 (매트 알파에 arg4 삭제 스트레인 (G418 : 매트, his3, ura3, leu2 및 arg4)를 교배에 의해 만들어진 his3, leu2, ura3 및 lys2) 및 meiotic haploids는 tetrad 절개로 얻은되었습니다. 따라서, 제어 변형은 라이신과 아르기닌을위한 auxotroph입니다. 미끼 스트레인은 ​​벡터 pBS1479를 사용하여 PCR - 중재 상동 재조합에 의해 만들어졌다. 따라서, 본 연구에서 사용되는 에피토프 태그 협동 친화 정화 (TAP) 태그 (3)입니다. </li></ul></li><li> SILAC : <ul><li> SILAC 제어 변형은 98% L - 라이신 - 4 ,4,5,5 - D4 (0.03 g / L, 캠브리지 동위 원소)와 98% L - 아르기닌 - 13 C 6 (와 보충 최소한 중퇴 미디어에 자란 0.036 g / L, 캠브리지 동위 원소). </li><li> 미끼 스트레인 정상 원소 아미노산과 함께 정기적으로 다양한 매체 성장했다. </li></ul></li><li> TAP 정화의 첫 단계 [이 프로토콜은 콜드 스프링 하버 연구소 물론 매뉴얼 (4)에서 적응이다] ** -80 ° C의 냉장고에서 정화, 사전 진정 박격포와 유봉 전날 밤. 또한, -20에서 사전 진정 비커 ° C ** 오른쪽 사용 (문화 1 패)을하기 전에이 버퍼를 만들기 : <ul><li> 25 μl PIC (1 / 2000), 1M DTT 50 μl 30 ML NP - 40 버퍼 </li><li> 2백분의 1 PIC, 1M DTT 50 μl와 함께 NP - 40 버퍼 20 ML </li><li> 2천분의 1 PIC와 함께 TEV - C 10 ML 버퍼, 1 M DTT 10 μl </li></ul></li></ol> SILAC 및 준비 <ol><li> 미끼 변형 및 컨트롤의 각 OD 600 = 1.0-1.3을 별도로 부담 1L 성장. </li><li> 500ml Nalgene의 원심 분리기 튜브의 문화를 하거라. 2580X G.의 부하와 펠릿 세포의 균형 </li><li> 50ml 원심 튜브에 세포를 전송하는 동안 차가운 DH 2 O의 50ml로 가만히 따르다 미디어 resuspend 펠릿. 펠렛 세포. ** 당신은 -80에서 펠렛을 멈추게 할 수도 ° C ** </li><li> ** 중요 ** 건조 펠렛 중량에 의해 1시 1분에서 미끼의 변형 문화 컨트롤 변형 문화를 일치합니다. </li><li> 1 / / 2000 테아제 억제제 칵테일 (PIC)와 NP - 40 버퍼 10ml 각 세포 펠렛을 Resuspend. </li><li> 직접 다른 하나의 튜브를 decanting하여 두 세포 문화를 섞는다. 연마 </li><li> 미리 차게 절구에 액체 N 2를 붓고 그것이 완전히 증발 수 있습니다. 원운동에서 박격포에 셀 2 ML을 추가합니다. 세포를 고정하기 위해 몇 가지 액체 N 2를 붓고. 세포가 미세 분말이 될 때까지 세포를 분쇄. 모든 세포가 지상 때까지 과정을 반복합니다. ** 세포 해동을 허용하지 마십시오. 액체 N 2 필요 ** 추가 </li><li> 실온에서 얼음처럼 차가운 비커와 해동에 분말을 전송합니다. 가장자리가 해동하기 시작하면, 2백분의 1 PIC와 함께 NP - 40 버퍼 20 ML를 추가합니다. </li><li> 40 ML Nalgene 튜브로 원유의 lysate를 전송합니다. 39,000 xgfor 30 분에 스핀. </li><li> 기다리는 동안, 세파 로스 2B 비즈 (GE) 300 μl을. NP - 40 버퍼 (1 / 2, 000 PIC) 3 번 300 μl의 구슬을 씻으십시오. 그들이 1:1 슬러리에 있도록 300 μl 버퍼에 비즈를 Resuspend. 사전 클리어링 세포 lysate </li><li> 새로운 튜브에 뜨는을 전송하고 세파 로스 2B 비즈를 추가합니다. 30 분 추운 방에 회전 플랫폼에 품어. </li><li> 기다리는 동안, IgG 세파 로스 6 구슬 (GE) 300 μl을. NP - 40 버퍼 (1 / 2, 000 PIC) 3 번 300 μl의 구슬을 씻으십시오. 그들이 1:1 슬러리에 있도록 300 _l 버퍼에 비즈를 Resuspend. </li><li> 펠렛 비즈. 새로운 튜브에 뜨는를 전송합니다. ** 서양 나중에 모래 바닥에 대한 세포 lysate의 나누어지는 저장하기 ** IgG 세파 로스 비즈에 바인딩 </li><li> 원심 분리기로 세파 로스 2B 비즈를 제거합니다. IgG 비즈를 (이전 1시 1분 슬러리에 준비) 추가합니다. 보다 효율적인 바인딩을위한 두 튜브로 내용을 구분합니다. 2 시간을 위해 추운 방에 회전 플랫폼에 품어. </li><li> 비즈를 스핀 다운. ** 언바운드 파벌의 나누어지는 ** 저장 </li><li> 10 ML NP - 40 버퍼 (1 / 2, 000 PIC)와 구슬을 씻으십시오. </li><li> 3 ML TEV - C 버퍼 (1 / 2, 000 PIC)와 구슬을 씻으십시오. ** 구슬 나누어지는을 저장합니다. 이것은 구속력이 **의 효율성을 반영됩니다 TEV 프로 테아제 절단하여 IgG 비즈에서 Eluting </li><li> TEV - C의 1ml 버퍼 (1 / 2, 000 PIC와 함께)에 AcTEV 5 μl를 추가합니다. 혼합 후,보다 효율적인 믹싱을위한 두 1.5 ML Eppendorf 튜브에 내용을 별도의. 밤새 추위 방에 회전 플랫폼에 품어. </li><li> 비즈를 스핀 다운하고 신선한 1.5 ML 튜브에 eluate을 전송하기만하면됩니다. TEV - C 버퍼의 추가 0.5 ML로 비즈 와시 </li><li> 한 튜브에 elaute을 결합합니다. 당신은 총 eluate 1.5 ML 있어야합니다.** TEV eluate의 나누어지는 ** 저장 Trichloroacetic 산 (TCA) precipatation은 (질량 분석계 기반 분석을 위해, 우리는 EtOH / 아세트산 석출를 사용하는 것이 좋습니다) </li><li> 백퍼센트 TCA와 TCA 25 % aliquots를 조정합니다. 이렇게하려면 750 μl 각 2 튜브에 1.5 ML의 eluate를 구분한다. 튜브에 TCA 100 % 250 μl를 추가합니다. 귀하의 솔루션은 밀키 설정해야합니다. </li><li> 정기 vortexing 30 분 얼음에 놓습니다. </li><li> 30 분 추위 방에 테이블 상단 원심 분리기의 최대 속도로 회전. </li><li> 최대 속도 (추운 방)에서 5 분 0.05 N HCL와 스핀이 들어있는 얼음처럼 차가운 아세톤 500 μl와 함께 한 번 씻으십시오. </li><li> 최대 (추운 방)에서 5 분 얼음처럼 차가운 아세톤과 스핀 500 μl와 함께 한 번 씻으십시오. </li><li> 조심스럽게 아세톤 건조 펠렛를 제거합니다. </li><li> 실버 얼룩의 경우, 1X SDS 샘플 버퍼의 50 μl의 펠렛을 resuspend. EtOH / 아세테이트 precipatation </li><li> 글리코겐 20 μg 추가 </li><li> 100 % EtOH로 5 배 희석하고 샘플을 2.5 M 재고, 산도 5.0 50 MM NaCH 3 COO로 조정 </li><li> 2 시간에 대한 솔루션을 스탠드 </li><li> 펠렛은 실온에서 12,000 X g에서 10 분 원심 분리하여 단백질을 시켰던. </li></ol> NP - 40 버퍼 (200 ML에 대한) <table border="0"><tbody><tr><td></td><td> 주식 농도 </td><td> 추가 </td></tr><tr><td> 10 MM 나트륨 인산 버퍼 (산도 7.2) </td><td> 0.1 M </td><td> 20 ML </td></tr><tr><td> 150 MM NaCl </td><td> 2 M </td><td> 15 ML </td></tr><tr><td> 1퍼센트 NP - 40 </td><td> 백퍼센트 </td><td> 2 ML </td></tr><tr><td> 50 MM NaF </td><td> 1 M </td><td> 10 ML </td></tr><tr><td> 0.1 MM 나 세 할머니 4 </td><td> 10 MM </td><td> 2 ML </td></tr><tr><td style="text-align:right;" colspan="3"> 200 ML에 볼륨 </td></tr></tbody></table> 사용하기 전에 ADD : <ol><li> 1 M DTT 1 / 1, 000 </li><li> PIC의 1 / 2, 000 (프로 테아제 억제제 칵테일) </li></ol> TEV - C 버퍼 (50 ML 용) <table border="0"><tbody><tr><td></td><td> 주식 농도 </td><td> 추가 </td></tr><tr><td> 25 MM 트리스 (산도 8.0) </td><td> 100 MM </td><td> 12.5 ML </td></tr><tr><td> 150 MM NaCl </td><td> 2 M </td><td> 3.75 ML </td></tr><tr><td> 0.1 % NP - 40 </td><td> 백퍼센트 </td><td> 50 _l </td></tr><tr><td> 0.5 MM EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 500 MM </td><td> 50 __l </td></tr><tr><td style="text-align:right;" colspan="3"> 50 ML에 볼륨 </td></tr></tbody></table> 사용하기 전에 ADD : <ol><li> 1분의 1, 1M DTT의 000 </li><li> PIC 1 / 2, 000 </li></ol>

<ol>
	<li>Loewen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+cortical+ER+in+yeast+is+required+for+normal+septin+organization.">Inheritance of cortical ER in yeast is required for normal septin organization.</a> Jour. Cell Biol. 179, 467-483 .</li><li>Ong, S., Foster, L. J., Hoog, C. L., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric-based+approaches+in+quantitative+proteomics.">Mass spectrometric-based approaches in quantitative proteomics.</a> Methods. 29, 124-130 .</li><li>Puig, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tandem+Affinity+Purification+(TAP)+method:+a+general+procedure+of+protein+complex+purification.">The Tandem Affinity Purification (TAP) method: a general procedure of protein complex purification.</a> Methods. 24, 218-229 .</li><li>Amberg, D. C., Burke, D. J., Strathern, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+Yeast+Genetics.">Methods in Yeast Genetics.</a> , 123-125 .</li></ol>

정화 절차를 수행하는 동안 저장된 aliquots (1) 사전 허가 세포 lysate, (2) 바운드 분수 (3) 언바운드 분율, 그리고 (4) eluted 소수를 포함해야합니다. 우리는 실험의 바인딩과 eluting 효율성을 반영하기 위해 안티 - TAP 항체 또는 실버 얼룩을 사용하여 모래 바닥 서양하여 위의 aliquots의 단백질 내용을 분석하는 것이 좋습니다. 은색 스테인드 젤과 얼룩의 예는 그림 1에 표시됩니다. SIL...

identification of protein complexes with quantitative proteomics in s. cerevisiae

Lipids 중요한 세포 신호 전달 분자로, 최근에는 장벽로서 세포 프로세스 compartmentalization에서 세포 세포막 그 함수의 빌딩 블록이며,. 그러나, 단백질과는 달리, lipids 작은 소수성 분자는 것을 주로 막 연락처 사이트 (MCSs)에서 발생하는 가상 아르 제대로 설명 nonvesicular 경로로 트래픽. MCSs는 endoplasmic reticulum (ER)가 제휴 organelle, 예를 들어, 플라즈마 멤브레인 (PM)와 직접적인 신체 접촉을 만드는 지역입니다. ER - PM MCSs의 ER 부분은 그 지질 합성이 해당 사이트로 이동하고 MCSs는 지질 트래픽 중요하다는 것을 뜻합니다 제안, 지질 합성 효소에 풍부합니다. 효모는 1000 셀 당 연락처 및 모든 eukaryotes에서 보존 자연과, 때문에 그들의 풍요의 ER - PM MCSs를 공부하는 이상적인 모델입니다. MCSs을 구성하는 단백질을 잠복하면 lipids 트래픽이 세포에 달성하는 방법 이해하는 것이 중요합니다, 그들은 신호 분자 역할을하는 방법. 우리는 Scs2p라는 ER은 ER - 오후 MCSs에 집중 발견과 형성에 중요합니다. 우리는 Scs2p의 분자 파트너 잠복에 초점을 맞추고 있습니다. 단백질 단지의 확인은 전통적으로 첫번째 질량 분광법에 의해 펩티드의 단백질 밴드와 분석에 - 젤 소화 다음 겔 전기 영동에 의해 정제된 단백질 샘플을 해결에 의존합니다. 이것은 종종 단백질의 작은 하위 집합에 대한 연구를 제한합니다. 또한, 단백질 단지는 절차를 수행하는 동안 denaturing 또는 이외의 생리 조건에 노출되어 있습니다. 이러한 문제를 회피하기 위해, 우리는 편견과 계량 데이터를 추출하기 위해 대규모 양적 proteomics 기술을 구현했습니다. 우리는 태그가 지정되지 않은 제어 변형에 단백질의 주요 동위 원소의 핵을 통합하기 위해 세포 배양에서 아미노산 (SILAC)와 안정 동위 원소 라벨을 사용합니다. 태그 문화와 태그가 지정되지 않은, SILAC - 레이블 문화의 동등 권 함께 혼합하여 액체 질소에 연삭에 의해 lysed 있습니다. 그러면 단백질 단지를 잡아 친화 정화 절차를 수행합니다. 마지막으로, 우리는 고성능 액체 크로마 토그래피 / 탠덤 질량 분석계로 분석을위한 준비가 단백질 샘플을 촉진. 가장 중요한 것은, 제어 변형에 단백질은 무거운 동위 원소에 의해 표시되고 미끼 변형에 레이블이 지정되지 않은 단백질에 대한 계량 수있는 질량 / 비용 변화를 생산합니다. 따라서, 오염 물질, 또는 unspecific 바인딩을 쉽게 제거하실 수 있습니다. 이 접근법을 사용함으로써, 우리는 ER - 오후 MCSs에 집중 여러 소설 단백질을 발견했습니다. 여기 우리는 우리의 접근 방법에 대한 자세한 설명을 제시한다.

Watch this Scientific Journal Video about Identification of protein complexes with quantitative proteomics in S. cerevisiae at JoVE.com

Identification of protein complexes with quantitative proteomics in S. cerevisiae

여기 Saccharomyces cerevisiae의에서 단백질 단지를 식별하기위한 새로운 양적 proteomics 기술을 설명합니다. 본 연구에서는, 우리는 높은 특이성과 ER 단백질 Scs2p의 바인딩 파트너를 식별하는 탠덤 질량 분석계 다음 친화 정화와 함께 SILAC 방법을 사용했습니다.

S. cerevisiae의의 양적 proteomics와 단백질 단지의 식별

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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13.1K 조회수.  University of British Columbia - UBC. 여기 Saccharomyces cerevisiae의에서 단백질 단지를 식별하기위한 새로운 양적 proteomics 기술을 설명합니다. 본 연구에서는, 우리는 높은 특이성과 ER 단백질 Scs2p의 바인딩 파트너를 식별하는 탠덤 질량 분석계 다음 친화 정화와 함께 SILAC 방법을 사용했습니다.

비디오: Identification of protein complexes with quantitative proteomics in S. cerevisiae

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

S. cerevisiae의의 접착 및 아가르 침략을위한 분석

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

연구

생물학

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

세계 S. cerevisiae의에서 organelle 형태를 연구하기 위해 높은 처리량 방법

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

지방산의 정량 Phosphoproteomics은 자극 Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

를 사용하여 연령에 의존 게놈의 불안정성 유학 S. cerevisiae 연대기 수명 모델

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

의 단백질 단지의 식별<em&gt; 대장균</em&gt; 직렬 질량 분석법과 함께 순차적 펩타이드의 동질 정화를 사용하여

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

의 흐름 세포 계측법 기반 정화<em&gt; S. cerevisiae</em&gt; Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

의 모니터링 단백질 접힘에 대한 결합 분석 실험<em&gt; 사카 cerevisiae의</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

식물 단백질 복합체의 번역 후 변형의 확인

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

SILAC - 면역 정량 프로테오믹스를 사용하여 포유 동물 세포에있는 단백질 상호 작용 파트너의 식별

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...