251

1436-1435

2

01

4

1

SDS-

PAGE

PAGE from

(Poly Achrelamyde Gel Electrophoresis)

(Lammli (1970:

2014 2

:

E. coli

24 LB 24

.

.

0.1 (Phosphate buffer)

(140mM NaCl; 2.7mM KCL; 10mM Na2HPO4;

1.8mM KH2PO4; pH 7.3 (PBS))

10 % Ammonium persulfate (NH4)2S2O8

4

MeOH90 %

2014 3

:

orthophosphoric acid 85%

DDW.

Coomassie Brilliant Blue G-250 dye

( ) Bradford Reagent.

Bromophenol blue

Glycin

SDS (sodium dodecyl sulphate )

Tetramethylethylenediamine (TMEDA or TEMED (CH3)2NCH2CH2N(CH3)2

2014

4

:

Tris base

methanol

acetic acid

glacial acetic acid

TCA Trichloroacetic acid (also

known as trichloroethanoic acid)

Chilled aceton

100 - 1) )

(./ )( ) /

.

2014 5

:

30% Acrylamide

Protein ladder (marker)

1 Whatman filter paper no.1

NaOH (01 )

100 .

vortex mixer

Cooling Microfuge.

2014 6

:

: :

Eppendorf 2.

2 15

.

7( 0.1 ) 1.5

. 10 / 10.000 4

SDS-PAGE

.western blotting

20

14

7

:BRADFORD ASSAY

(.Bradford (1976

( ) ) ( )

. (

:

-1 .

-2 .

( )

.

. 595 .

2014 8

: -:

:Bradfords ReagentX 5

100 % 90 50

orthophosphoric acid 85.%

100Coomassie

Brilliant Blue G-250 dye

.DDW 1

1 Whatman filter

paper no.1 . pH -0.01.

. 4

1) 1:4 X )

DDW pH 1.1.

2014 9

( )

) LYSOZYME

(

2014 10

:

BRADFORDS REAGENT

TCA ( 10 %

. 10 / 5000 (

10 / 3300 .

1

. NaOH 01

20

14

11

:

BRADFORDS REAGENT

20

14

12

BSA

BOVINE SERUM ALBUMIN STANDARD CURVE

BSA 1 .

/ 50 40 30 20 10 0 .

Quartz cuvette ( 1.4:)

0.2

0.8

595

.O.D

.

2014 13

BSA

BOVINE SERUM ALBUMIN STANDARD CURVE

20

14

14

:

.SDS-PAGE

2

01

4

15

: -:

:

1 2.5 Tris HCl (pH=6.8 )

b-mercaptophenol 2.5 SDS 1

.DDW 40

:

5.8 5

10 10

.DDW

2014 16

17

20

14

: : :

Electrode buffer:

3 Tris base 1 14.42SDS 1000 DDW.

:

1 Coomassic brilliant blue R 180 180 DDW 40

.

:

400 100 MeOH 400DDW .

.magnetic stirrer

2014 18

19

20

14

: : :

:

%12A % 4B

2014 20

A

(ml)

B

(ml)

H2O 9.90 6.80

30% Acrylamide 12.00 1.70

1.5 M Tris (pH 8.8) 7.50 -

1.0 M Tris (pH 6) - 1.25

10% SDS 0.40 0.10

10% APS 0.40 0.10

TEMED 0.016 0.12

:

:

A B .well

10 .tracking dye

.

8 bands

.

marker

20

14

21

20

14

22

SDS-PAGE SDS-Polyacrylamide Gel Electrophoresis

2

01

4

23

SDS-PAGE(POLYACRYLAMIDE GEL

ELECTROPHORESIS)

.

DNA RNA.

.

SDS .

SDS .

(. anode )

molecular sieving (. )

bands

24

20

14

( BIO-RAD) :

( )

.

25

20

14

.

casting frame

.

26

20

14

.

27

20

14

: Gel preparation :

TEMED APS .

28

20

14

Resolving Gel

10%

Stacking Gel

4.5%

5.65 DDW 4

1.65 3.3 %30

Tris pH 8.81.5M 2.5 ---

1M Tris pH 8.8 --- 2.5

SDS10% 0.1 0.1

10%

Ammonium

persulphate

(APS)

0.1 0.1

TEMED 0.004 0.004

resolving

. 1

29

20

14

.

30- 20 .

30

20

14

stacking gel

APS TEMED . .

.

stacking gell

31

20

14

.

10 .

32

20

14

: 1: 4

30 37 .

33

20

14

RUNNING THE GEL

wells .

.

.

Bio-Rad tank

10 X SDS running buffer (pH 8.3)

.

well. 34

20

14

.

.

1 180 .

.

35

20

14

( )

36

20

14

.

30 .

8 .bands

. protein markers

SDS-PAGE.

.

Acrylamide . neurotoxin

37

20

14

SDS-PAGE

38

20

14

(SDS (SODIUM DODECYL SULPHATE

.

(denature )

) poly peptide

primary structure .)

-SDS

PAGE.

. Protein ladder

39

20

14

40

20

14

HT

TP

://WW

W.J

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E.C

OM

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PA

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41

20

14

http://www.youtube.com/watch?v=EDi_n

_0NiF4

http://www.youtube.com/watch?v=XUjLO

-ek2C8

../../Videos/RealPlayer Downloads/How to Run an SDS-PAGE gel3.flv

42

20

14

../../Videos/RealPlayer Downloads/How to Stain an SDS-PAGE gel2.flv../../Videos/RealPlayer Downloads/SDS PAGE.mp4../../Videos/RealPlayer Downloads/SDS-PAGE (polyacrylamide gel electrophoresis)3.meta

43

20

14

PAGE of rotavirus proteins stained with

Coomassie blue

Silver stained SDS

Polyacrylamide gels

http://en.wikipedia.org/wiki/Rotavirus