基因分子生物学实验：2. EMSA

1、EMSA electrophoresis mobility shift assay Original articles about EMSA 1. Heterogeneity of chromatin subunits in vitro and location of histone H1. Nucl Acids Res. 1976;3:477492. 2. Gel electrophoretic separation of transcription complexes: an assay for RNA polymerase selectivity and a method for pro

2、moter mapping. Nucl Acids Res. 1979;7:18511867. 3. Equilibria and Kinetics of Lac Repressor-Operator Interactions by Polyacrylamide Gel Electrophoresis. Nucl Acids Res. 1981;9:6 5056525. 4. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to c

3、omponents of the Escherichia coli lactose operon system. Nucl Acids Res. 1981;9:30473060. Similar to current buffer PAGE to characterize protein-DNA complex Progress of EMSA Biotinylated probes in the electrophoretic mobility shift assay to examine specific dsDNA, ssDNA or RNA-protein interactions.

4、Nucleic Acids Res. 1995, 25;23(18):3792-3. Chemiluminescence-based electrophoretic mobility shift assay of heparinprotein interactions Analytical Biochemistry, 2006, 349(1):156-158 EMSA whole process nuclear extraction P32 label oligonucleotides PAGE preparationbinding reaction loading, electrophore

5、sis order gamma- P32 ATP Anneal oligonucleotides de-assemble apparatus, autoradiograph, exposure Synthesize oligonucleotides （20bp左右） P53蛋白 DNA结合元件 Preparation 1: order P32 ATP P32 with half life 15days, order on time order by specialist gamma-P32 ATP, or gamma-P32 dATP not alpha-P32 p32 use alpha-P

6、32 to fill in sticky ends Preparation 2：design and synthesize oligonucleotides design wild type probe mutant probe synthesize probe specially modified / not modified from literature mutate: least number bases and most conserved bases (突变：嘌呤和嘧啶互 变，较多使用A-C, T-G互变) 生物素EMSA: biotin modified in 5 or 3 同位

7、素EMSA: not modified Preparation 2：anneal oligonucleotides 10 x annealing buffer：200 mM Tris 8.0, 10 mM EDTA 8.0, 500 mM NaCl dissolve single strand probe to a concentration of 25uM 50 reaction system：25uM single strand probe 20ul, 10 xannealing buffer：5ul,H2O 5ul 。 put mineral oil on surface,boil 5

8、min,cool in water naturally, about 34 or final concentration of probe 10uM store in -20 not in 4 (easy to degrade) avoid repeated freeze-thaw Preparation 3: nuclear extraction (NP40 method) 107 cells PBS two times gently resuspend in 1 ml buffer(10 mM HEPES pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGT

9、A; 1 mM DTT; 0.5 mM PMSF, cocktail) ice, 15min add 62.5 ul 10% NP-40 vigorously vortex, 10 sec 12000g, 30sec resuspend pellet in 200ul ice cold buffer (HEPES pH 7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM DTIT; 1 mM PMSF, cocktail) vigorously rock at 4 for 15 min 12000g,5 min determine protein conce

10、ntration store at -80 final concentration: 0.625% quickly ! about 515 ug protein extract or 5-25ng purified protein in a binding reaction about 500750g per 107 cells pierce NE-preparation method is similar to this method 破碎细 胞膜 离心沉淀 细胞核 对细胞 核进一 步操作 Dounce homogenization proper specification (gap siz

11、e) Check materials and apparatus Materials: Apparatus gamma-P32 ATP annealed oligos (modified/not modified for biotin-EMSA, labeled/unlabeled for P32-EMSA) nuclear extract proteins 5x TBE(54 g Tris base, 27.5 g boric acid,20 ml 0.5M EDTA (pH 8.0) T4 polynucleotide kinase(Takara or NEB) poly dI/dC or

12、 salmon sperm DNA(ssDNA) (1g/l，aliquot 50100 l，- 20 ) 10%BSA 29:1% acrylamide:bisacrylamide 0.1% bromophenol blue X ray film antibody electrophoresis glass pate(2pieces) plunge power 37 centi 65 centi monitor Step 1: label oligos 25ul reaction system: oligos (10uM): 2ul -32P ATP: 2ul 10 x T4 PNK buf

13、fer: 2.5ul H2O: 17.5ul T4 PNK enzyme(6-10U/ul): 1ul spin 37 , 30-60min 65 , 10 min to inactivate T4 PNK enzyme 1ul for new P32 and 2ul for old P32 4 , storage protection from P32 protecting tube wall from P32 5-PO3-TCAAAGTACA-OH-35-PO3-TCAAAGTACA-OH-3 T4 PNK Step 2: binding reaction final con.storag

14、e con.volume 50mM1M Tris pH 7.550l 750mM2M KCl 375l 1mM100mM DTT10l 1mM0.5M EDTA5l 50%100% glycerol500l water60l prepare 5x binding buffer freshly important: different binding buffers (nuclear, not cytosolic) 5binding buffer 2l 10%BSA 1l ssDNA (1g/l） 1l -32PATP probe 1l nuclear extract 5g x ul water

15、 to 10l 10 ul binding reaction system 37, 30min 515g crude extract,5-25ng purified protein lastly add to prevent degradation if using Ab, firstly incubate Ab RT for 15-20min, then add probe and incubate further for 30min glycerol concentration may vary: 515% Step 3: PAGE preparation and pre- electro

16、phoresis 10 ml 5% PAGE 30% A solution 3.3ml 5TBE 2ml 50% glycerol 1ml ddH2O 13.7ml 10%APS 100l TEMED 10l flush loading wells pre-electrophoresis: 100V,60-90min。 not precipitated prepared freshly important necessary Step 4: electrophoresis and exposure add 2l 0.1% blomophenol blue to each sample, mix

17、 electrophoresis: 150V(5- 10mA) 2-2.5hours 0.1% blomophenol blue without mixed with binding buffer run and blomophenol blue mixed with binding buffer run near 1/3-1/2 from top use an additional well for 0.1% blomophenol blue only to indicate electrophoresis front line disassemble apparatus protectio

18、n human and water pool, desk from P32 contamination put gel in X ray box with X ray film about 8-12 hours for P32 in day 1 Principle of EMSA HNF4 binds to HNF4 site in EMSA experiment unspecific band binding supershift HNF4 site IIHNF4 site I 1 2 3 4 1 2 3 4 1 2 3 1.wt probe 2.wt probe+ myc-HNF4 3.w

19、t probe+myc-HNF4 + myc-Ab 4.mutant probe+myc-HNF4Positive control (ApoCIII) EMSA experiment using nuclear extract from 293T cells. Quantify TF-DNA interaction in EMSA EMSA can also be used for quantification of interaction of TF with DNA: Protein ligands Factors influencing interaction of protein wi

20、th DNA Modification of proteins: Phosphorylation Zhang Ran, 2006 Biotin labeled EMSA Advantage: safety and convenience 1. short time and easy handling 2. specialized apparatus: not confused with Western to prevent SDS contamination Disadvantage: high background 1.wide existence of biotin in environm

21、ent 2.unspecific binding of proteins to probe Biotinylated probes in the electrophoretic mobility shift assay to examine specific dsDNA, ssDNA or RNA-protein interactions. Nucleic Acids Res. 1995, 25;23(18):3792-3. Original paper: Product: LightShift Chemiluminescent EMSA Kit pierce 1.Purify nuclear extract 2.Optimize binding buffer to diminish unspecific binding shift supershift Protection from radioactivity radiation (by large atoms 100): He2+ radiation: electronic emission radiation/X ray: photon Inspection by Monitor: alpha, beta, gamma NOT