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Electrophoresis

Electrophoresis

Arne Tiselius(1959), Electrophoresis Theory, Methods and Applications.

1809Ress1937Tiselius Tiselius A121948

1940 60 1967Shapiro SDS 1969WeberSDS- 1970LaemmliSDS

701975OFarrell ISO-DALT1985Gorg IPG-DALT

90

1(electrophoresisEP)

2(electrophoretic migration)EMcm 3(migration time)tmin 4(electrophoretic velocity)V cm /min5(electric field strength)E V/cmE = V /LVL

AB6(electroosmosis)

7(relative mobility)mR

(cm)mR = (cm)

pH +-

Q F=E Q 1,F F=6rV F=F EQ= 6rV

,(V)(E)(Q),(r)()DNA234

4 m m = V/E = Q/6r 5

4 m

pHUm U = m 6 65 U = 7

.Q.6r 7QpH

U

r; E q 1

2E (1)E=2-10v/cm (2) E=70200Vcm

3pHpH0.02O.2

4

5Q=I2RT

6

123

12(1)(2)(3)

:(1). (2)(molecular sieving effect)3

1(1)(600V)SDS-(2)(3000V)DNA(3)(30000-50000V)

21Tiselius

2(disc electrophoresis)

3(slab electrophoresis)

3

1 D-36L- 10045

2

3DNADNA

DNA200bpDNA; 5500bp; 1bpDNADNA

1 DNA()DNA20kb1DNA

2 DNA

DNA DNA>DNA>DNADNA()O(Rm=O)DNA()(Rm>O)3

456

TAE 50TBE 5 TPE 10 2()3

TAEDNATAE1013 kb DNATAEDNATAETAETrisEDTA

TBETrisEDTATBE1 kbDNA

DNATPETrisEDTA

6O.25-0.25%FF-40%4O.25-0.25%FF-15%m/V)400O.25-0.25%FF-30% 4 TBE4

5(ethidium bromideEB)EB(Ag+)Ag+EB200.DNA

X-ray structure of a complex of ethidium with 5-iodo-UpA

612DNA

712345

1.

4M1M 0M

0.1 0.25 0.5 1.0 5.0 DNA Hind III fragments (g)Effect of sample DNA concentration on resolution.

12

12PAGE3SDS-PAGE4pH 56DNA7

60DavisSDS-

1 ()(1)(100500 V) 210 Vcm(2)(200010000 V) 50200Vcm 2pH pHpHpH(pI) 3 O.02O.2(ionic strength) I= 1/2 CZ2 Ic(molL)z O.15 mo/L Na2SO4 I=1/20.15212+0.1522=0.45

2pH pHpHpH(pI)

OH- pH

5 6

123

D-36L-10045

1%150nm (9899)

1(1)DNA DNA()DNA20kbDNADNADNA

(1) ()12 mmEDTA(pH8.0)Tris-(TAE)Tris-(TBE)Tris-(TPE)50 mmolL(pH7.57.8)TAETBE510O.5

TAE(Tris)TAETrisEDTATAE(TBE)DNA1013 kbTAEDNATAETAE()

TBE(Tris-)TrisEDTATBETBE1 kbTBEDNA

Effect of salt concentration on resolution of fragments.Samples in all three lanes are identical except for [salt].