Protein DNA RNA extraction Protein Trypsin Digestion 02

생물학연구를 위한 최적의 시스템 안내 Protein , DNA, RNA extraction Protein Trypsin Digestion ㈜씨엠코퍼레이션에이티 전화 02. 451. 9001

Old Method New Method 13 US patents 4 EU patents 1 AU patent 열발생, 단백질 변성, 오염, Low Quality, Open Method 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

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

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

Pulse Tube Denaturation of Nucleic Acids In cr ea sin g Pr es su r e Denaturation of Proteins (monomeric) Disassociation of Complex Structures (multimeric) Disruption of Viruses DP Killing of Cells, Bacteria, Fungi High Pressure에 의한 Pulse Tube의 Lysis Disk를 통한 Powerful한 시료 파쇄 1 Lysis buffer 2 a a Sample b b 3 a c b Pulse Tube Pressure Applied 4 5 a a c b b

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

결과비교 Murine Liver Proteome: PCT, sonication, and ground glass tissue grinder sonicator lysate PCT lysate 1, 739 spots 2, 126 spots ground glass tissue grinder lysate 1, 853 spots 10 cycles of 20/20 s at 35, 000 PSI/atmospheric pressure IPG p. H 4. 5 -6. 5, Second dimension: 6 -15% precast gels

Escherichia coli lysis by PCT or bead mill PCT (35, 000 psi, 5 X 20 seconds) Total spot volume: 6569661 (+14. 2%) Number of spots detected: 801 (+5. 4%) BEAD MILL (1, 800 oscillations min-1, 3 X 30 seconds) Total spot volume: 5751701 Number of spots detected: 760

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

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

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

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

Extraction Kit 소개 2 SB Single sample에서 Protein, DNA, RNA를 모두 추출 Place sample into PULSE Tube. Add Reagent A +/- Reagent B. Vortex 10 -20 seconds Place into Barocycler and run program Remove from Barocycler and Vortex 10 -20 seconds. Transfer entire contents of PULSE tube to centrifuge tube and spin at ~12, 000 g for 10 min. Nucleic Acids Solid material contains RNA, DNA and some DNA-associated proteins. Proteins Lipids Upper non-polar phase contains extracted lipids. Dissolve pellet material in appropriate reagent for DNA or RNA isolation. Vortex sample and proceed with isolation according to manufacturer’s protocol. No liquid nitrogen grinding or other tissue disruption is necessary. GC-MS, LC-MS, TLC, NMR Lower liquid phase contains dissolved sample proteins. Remove solvent by evaporation or precipitation. SDS-PAGE, 2 D PAGE, Western blotting, LC-MS/MS, MALDI-TOF, etc. . .

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

Application for Trypsin Digestion

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

Customers: Frank Witzmann Myra Robinson Rosalind Rosenthal 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 HSPH Alexander Ivanov Elena Chernokalskaya Sunny Tam Douglas Hinerfeld • Jennifer Isbister • James Willett • Emmanuel Petricoin • Lance Liotta • Valerie Calvert