생물학연구를 위한 최적의 시스템 안내 Protein , DNA, RNA extraction

Presentation on theme: "생물학연구를 위한 최적의 시스템 안내 Protein , DNA, RNA extraction"— Presentation transcript:

1 생물학연구를 위한 최적의 시스템 안내 Protein , DNA, RNA extraction
Protein Trypsin Digestion ㈜씨엠코퍼레이션에이티 전화

2 Sample preparation in the Era of “analytical arms race”
가장 이상적인 시료 전처리 시스템의 조건 지질층과 molecular complexes가 잘 분리되야 함, but 단백질, DNA, RNA의 공유결합에 영향을 주면 안됨. 시료 전처리 중 한결같은 에너지가 제공되어야 함. 지질 단백질, 핵산의 구분이 용이하여야 함. 추출 buffer에 친화적이어야 함. Cross-contamination을 방지할 수 있어야 함 진행되는 동안 안정적으로 시료 enclosed 유지하여야 함 정확하게 온도를 제어하여야 함 Frozen samples을 즉시 적용가능해야 함 분석을 위한 실험방법과 잘 matching 되는지를 충족시켜야 함. Well-defined experimental goal and well-prepared sample are the foundation of success.

3 세포, 동물조직, 식물체, 바이러스 등 모든 시료 적용 시료 파쇄/추출/Proteolysis in one system
Old Method New Method 13 US patents 4 EU patents 1 AU patent Closed Method, 5분 이내 완료 세포, 동물조직, 식물체, 바이러스 등 모든 시료 적용 시료 파쇄/추출/Proteolysis in one system 단백질 변성 없음, High Quality 열발생, 단백질 변성, 오염, Low Quality, Open Method

4 Multi-stage extraction approach employing orthogonal methods
Extraction 100 mg tissue: 1200 μL of solvent supernatant centrifugation pellet resuspend in appropriate buffer 50 μL for protein assay 250 μL for 2DGE SDS PAGE 200 μL for dot blot 2nd Extraction Exchange solvent if necessary centrifugation pellet Supernatant* etc. no reducing agent reduction alkylation ultrafiltration DTT no reduction no detergent 50 μL for protein assay 250 μL for 2DGE 50 μL for SDS PAGE 20 μL for dot blot * exchange solvent if necessary PRIMARY ANALYSIS SECONDARY ANALYSIS

5 Pressure Cycling Technology (PCT) 이란?:
“Hydrostatic High Pressure Cycles에 의한 시료 파쇄 biomolecular interactions의 정확하고 안정적인 제어”

6 1 5 4 3 2 a b c Pressure Applied Increasing Pressure DP Pulse Tube
Denaturation of Nucleic Acids Denaturation of Proteins (monomeric) Disassociation of Complex Structures (multimeric) Disruption of Viruses Killing of Cells, Bacteria, Fungi Increasing Pressure DP High Pressure에 의한 Pulse Tube의 Lysis Disk를 통한 Powerful한 시료 파쇄 1 5 4 3 2 a b c Pressure Applied Lysis buffer Sample Pulse Tube

7 PCT applications Human/Animal Tissue Fungi Virus Insects
Cultured Cells Plant Tissue Environmental Samples Forensic Samples Food Samples Microorganisms Homogenization Extraction Metabolomics DMPK Protein Purification DNA and RNA Purification Gene Expression RT-PCR qPCR Protein Refolding Immuno- diagnostics Food Safety Forensic Analysis Pathogen Inactivation Environmental Analysis

8 결 과 비 교 sonicator lysate 1,739 spots PCT lysate 2,126 spots
Murine Liver Proteome: PCT, sonication, and ground glass tissue grinder sonicator lysate 1,739 spots PCT lysate 2,126 spots ground glass tissue grinder lysate 1,853 spots 10 cycles of 20/20s at 35,000 PSI/atmospheric pressure IPG pH , Second dimension: 6-15% precast gels

9 Escherichia coli lysis by PCT or bead mill
(35,000 psi, 5X 20 seconds) Total spot volume: (+14.2%) Number of spots detected: 801 (+5.4%) BEAD MILL (1,800 oscillations min-1, 3X 30 seconds) Total spot volume: Number of spots detected: 760

10 Tests for arthropod-borne pathogens
Ixodes Scapularis DNA Borrelia burgdorferi DNA

11 A Dissolution Reagent Extraction Kit 소개 1 B Partitioning Reagent
PCT Dependent Detergent-Free Extraction of Proteins from Lipid-Rich Samples A Dissolution Reagent B Partitioning Reagent C Precipitation Reagent 12 PULSE Tubes™

12 kit performance Sonication PBS ProteoSolveLRS 2% SDS
200 mg of porcine adipose tissue per tube Sonication 2% SDS PBS ProteoSolveLRS 1 2 3 4 Nearly complete dissolution Clearly visible interface, Two liquid phases,

13 kit reproducibility IEF sample buffer A B 2 3 1
Murine Abdominal Fat Pad Protein Extraction B 1 2 3 Six 200 mg pieces of tissue (A-F above) were processed in parallel, and loaded in triplicate onto 8-16% gradient SDS-PAGE gel. Proteins from murine adipose tissue A: PCT + (9M urea, 4% CHAPS, pH=8) versus B: PCT + detergent-free ProteoSolve LRS

14 Extraction Kit 소개 2 SB Single sample에서 Protein, DNA, RNA를 모두 추출
Place sample into PULSE Tube. Add Reagent A +/- Reagent B. Vortex seconds Place into Barocycler and run program Remove from Barocycler and Vortex seconds. Transfer entire contents of PULSE tube to centrifuge tube and spin at ~12,000g for 10 min. Nucleic Acids Proteins Solid material contains RNA, DNA and some DNA-associated proteins. Lipids Lower liquid phase contains dissolved sample proteins. Remove solvent by evaporation or precipitation. Upper non-polar phase contains extracted lipids. Dissolve pellet material in appropriate reagent for DNA or RNA isolation. GC-MS, LC-MS, TLC, NMR SDS-PAGE, 2D PAGE, Western blotting, LC-MS/MS, MALDI-TOF, etc... Vortex sample and proceed with isolation according to manufacturer’s protocol. No liquid nitrogen grinding or other tissue disruption is necessary.

15 결 과 비 교 Protein, RNA and DNA Recovery From Rat Tissues SB
Brain Cardiac Adipose Liver Kidney MWS Br Ca Ad Li Kd Br Ca Ad Li Kd E P E P E P E P E Solvent removed by: E – evaporation; P - precipitation

16 Application for Trypsin Digestion

17 In-Gel & Solution application을 위한 전용 MicroTube
20분 이내 Proteolysis 기존 overnight 뛰어난 효율성 In-Gel & Solution application을 위한 전용 MicroTube Bench-Top 형태의 자동화 시스템 정확하고 효율적인 Peptide cleavage Protein Modification이 전혀 없음 Sample Loss 최소화 Mass Spectrometry analysis를 위한 시료 전처리

18 Customers: HSPH Ric Schumacher Nathan Lawrence Gary Smejkal Chunqin Li
Jim Behnke Feng Tao Vera Gross Ilyana Romanovsky Ada Kwan Vernon Reinhold Dibya Himali Andrew Hanneman Sue Chase Frank Witzmann Myra Robinson Rosalind Rosenthal HSPH Jennifer Isbister James Willett Emmanuel Petricoin Lance Liotta Valerie Calvert Alexander Ivanov Sunny Tam Douglas Hinerfeld Elena Chernokalskaya