Download
1 / 11
110 likes | 335 Views
生命科學實驗. 生命科學實驗. Plasmid DNA miniprep . PCR R. E. digestion Gel purification, Ligation, Transformation Preparation of competent cells. Gene Plasmid b.p . ORF E.coli strain. Recombinant DNA Techniques (DNA Cloning). GST-LEU2 SDS-PAGE Coomasie Blue ( Green Angel) Staining
E N D
生命科學實驗 Plasmid DNA miniprep. PCR R. E. digestion Gel purification, Ligation, Transformation Preparation of competent cells Gene Plasmid b.p. ORF E.coli strain Recombinant DNA Techniques (DNA Cloning) GST-LEU2 SDS-PAGE Coomasie Blue (Green Angel) Staining O.D.280, Bradford and Bicinchoninic acid assays (BCA) Expression, Purification and quantification of recombinant protein M.W. Ab Detection of recombinant protein Immunological (Western) Analysis Application of recombinant protein Enzyme assay (DHase) of GST-LEU2 GST activity
Restriction Enzyme Check of pGEX-LEU2 Plasmid 637 1095 EcoRI LEU2 pGEX-5’ pGEX-2T pGEX-3’ BamHI SmaI EcoRI Uncut EcoRI EcoRI/BamHI V V V pGEX-LEU2 #3 pGEX-LEU2 #3 pGEX-LEU2 #3 pGEX-leu2 pGEX-leu2 pGEX-leu2 #1 #1 #1 #2 #2 #2 #3 #3 #3 5000 4000 3000 2500 2000 1500 1000 750 500 250
Outline for expression and purification of GST-fusion protein Clone gene into pGEX vector Test expression of fusion protein (small scale), SDS-PAGE Week 1 Week 2 Culture (large scale), harvest and lyse cells GST-fusion protein purification, making new protein gel for week 4 Week 3 Week 4 Detection and quantification of GST fusion protein
SDS在蛋白質表面均勻敷上一層負電荷 SDS為ㄧ介面活性劑，其非極性端會愈蛋白質結合，破壞蛋白質之二及結構時其變性，成為一線狀分子並且均勻帶上一層負電荷 原態蛋白質 6
Discontinuous electrophoresis improves resolution of protein separation Running (separating) gel: longer, with high pH (8.9). For resolving protein samples. Stacking (spacer) gel: shorter, with lower pH (6.8). For causing SDS-protein complex as stacks of narrow bands.
Expression of GST-LEU2 Protein GST GST-LEU2 GST-leu2 IPTG (h): 0 0 0 0 0 0 3 3 3 3 3 3 5 5 5 5 5 5 Green Angel Protein Staining 170 GST GST-LEU2 GST-leu2 130 IPTG (h): 95 72 170 55 130 43 95 WB: R anti-GST (1:3000) 34 72 26 55 17 43 34 26 17 20121108 SCH lab
GST specific activity: (△A340)/min × V (ml) × dilution = umol/ml/min εmM× V enzyme (ml) εmM(mM-1cm-1): the extinction coefficient for CDNB conjugate at 340 nm, for test in 1 ml cuvette = 9.6 mM-1 (path length -1 cm). V : the reaction volume, for test in 1 ml cuvette = 1 ml V enzyme: the volume of the enzyme sample tested = 0.01 ml 0.1 * 1* 1 Input: = 1.04 9.6 *0.01 0.4 * 1* 1 = 4.16 Purified 9.6 *0.01
0.1 * 1* 1 Input: = 1.04 9.6 *0.01 0.4 * 1* 1 = 4.16 Purified 9.6 *0.01
