Polyacrylamide Gel Electrophoresis (PAGE)

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POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)

Last Update: December 2006



PREPARE SOLUTIONS



1. 30% Acrylamide/ 0.8% Mix 90 g of Acrylamide, 2.4 g of N'N'-bis-methylene-acrylamide, and 300 mL of dH2O (filter through a 0.45m filter) Bis (300 mL): (Store at 4oC in a dark bottle)



2. 10% SDS (100 mL):

Weigh 10 g of SDS and add dH2O to 100 mL

3. 1X SDS-running buffer Mix 10 mL of 10% SDS, 100 mL of 10X Running buffer, and 890 mL of dH O 2 (1 L): 4. 10X Running buffer (5 Mix 151 g of Tris base, 720 g of Glycine, and dH2O to 5 L (Store at 4oC) L): 5. 1X Native buffer (1 L): Mix 100 mL of 10X Running buffer with 900 mL of dH2O 6. 3X Native Sample Buffer (100 mL):

Mix 30 mL of Glycerol, 18.8 mL of 1 M Tris-HCl, pH 6.8, 1.5 mL of 1% Bromophenol blue, add dH2O to 100 mL (store at room temperature)

7. 10% Ammonium persulphate (APS) (10 mL):

Mix 1 g of APS with 10 mL of dH2O (Aliquot and store at -20oC in the dark)

8. 1.5 M Tris-HCl, pH 8.8 Mix 182 g of Tris base, 700 mL of dH2O, pH 8.8 with 1N HCl, make to 1 L with dH2O (filter through a 0.45m filter) (1 L): (Store at 4oC) 9. 1 M Tris-HCl, pH 6.8 (500 mL):

Mix 60.7 g of Tris base, 300 mL of dH2O, pH 6.8 with 1N HCl, make to 500 mL with dH2O (filter through a 0.45m

10. 4X SDS-Sample Buffer (80 ml):

Mix 32 mL of Glycerol, 24 mL of 1 M Tris-HCl, pH 6.8, 16 mg of Bromophenol blue, 8 g of SDS, make to 80 mL with

filter) (Store at 4oC) dH2O and 20 mL of b-Mercaptoethanol (Aliquot and store at -20oC) (long term storage: -80oC)

11. 0.5% Agarose (100 Mix 0.5 g of agarose with 100 mL of dH2O mL): 12. 1% Bromo-phenol Prepare 10 mg /mL in dH2O blue: 13. 50% Glycerol (100 Mix 50 mL of glycerol with 50 mL of dH2O (Autoclave) mL): NOTE: running buffer can be replaced with 25 mM Tris- 0.2 M Tricine NOTE: f or native gels at lower pH buffer (~4-5), polymerize gel with 0.01% riboflavin, and shine light for polymerization (TEMED is not active at low pH) NOTE: keep 10% APS and 3X SDS Sample buffer at -20oC NOTE: keep Tris solutions at 4oC PROCEDURE 1. Seal Homemadegels with 0.5% agarose before pouring gel Prepare the acrylamide running gel as follows (all measurements in mLs): 10 %

Gel concentration

12.5%

(1/2) (1) (1/2) (1)

Number of gels

(2)

(3)

(4)

(5)

4

8

3.13 6.25 12.5 18.75 25 31.25

1.5 M Tris-HCl, pH 8.8

2.5

5

2.5

5

10

15

20

25

10% SDS

0.1

0.2

0.1

0.2

0.4

0.6

0.8

1.0

30% Acrylamide/0.8% Bis

3.3

6.6 4.17 8.33 16.66

25

33.3 41.65

10% APS

0.1

0.2

0.6

0.8

1.0

TEMED

0.01 0.02 0.01 0.02 0.04

0.06 0.08

0.1

dH2O

10

Total volume

0.1

20

0.2

10

0.4

20

40

60

80

100

Amounts in table (equivalent for 1 gel): 0.75 and 1.5 mm BioRad minigels = (1/4); BioRad 1.5 mm supergels = (2); Homemadegels 1.5 mm X 15 cm X 16 cm = (1) 2. Overlay ~1 mL of dH2O, isobutanol, or 2-propanol on top of the Running (lower) gel 3. After polymerization (~45 mins) remove the isobutanol/dH2O wash several times with dH2O and pour the Stacking (upper) gel Prepare the acrylamide upper gel (4%) as follows (all measurements in mLs): (1/2)

Number of gels

(1)

(2)

(3)

(4)

(5)

dH2O

3.6

7.2 14.4 21.6 28.8 36

1 M Tris-HCl, pH 6.8

0.63 1.25 2.5 3.75

10% SDS

0.05

30% Acrylamide/0.8% Bis

0.66 1.34 2.68 4.0 5.36 6.7

10% APS

0.05

TEMED

0.005 0.01 0.02 0.03 0.04 0.05

0.1

0.1

5

Total volume

0.2

0.3

0.2

10

0.3

20

5

6.25

0.4

0.5

0.4

30

40

0.5

50

4. Running condition: 1. Homemadegels: constant voltage; start at 100 V (not over 60 mA per gel). Once dye enters lower gel increase voltage to 150V. Running time 4-6 h. 2. Minigels: constant voltage; start at 60-70 V, once dye enter lower gel increase volt to 80-90 V Note: check there are no bubbles between the gel and the buffer Other running gel concentrations: Acrylamide gel concentrations determine the range of separation of proteins: Acrylamide concentration (%)

Linear range of separation (kD)

15

12-43

10

16-68

7.5

36-94

5*

57-212

*NOTE: be very careful when working with low acrylamide concentration gels since they are extremely delicate NOTE: the ratio of bisacrylamide to acrylamide also determines the size of the pores of the gel, but this is more empirical. A 1:29 ratio has been shown to resolve polypetides wich differ in size by only 3%. Running time and gel size also have much to do with separation, but for the most part as the front runs out of the gel, little change in separation occurs. Gel concentration

5%

Number of gels

(1)

(2)

(1)

(2)

(1)

(2)

(1)

dH2O

7.8

15.6

4.5

9

9.5

19

11.1 22.2

50% Glycerol

0.2

0.4

0.2

0.4

0.2

0.4

0.2

0.4

5

10

5

10

5

10

5

10

0.2

0.4

0.2

0.4

0.2

0.4

0.2

0.4

30% Acrylamide/0.8% Bis 6.6

13.3

10

20

5

10

3.3

6.6

10% APS

0.1

0.2

0.1

0.2

0.1

0.2

0.1

0.1

TEMED

0.01 0.02 0.01 0.02 0.01

3 M Tris-HCl, pH 8.8 10% SDS

20

Total volume

10% 10% 15% 15% 7.5% 7.5% 5%

40

20

40

(2)

0.02 0.01 0.02

20

40

20

40



PROCEDURE - SDS/NATIVE GRADIENT PAGE NOTE: amounts (equivalent for 2 gels since only half is used per gel): 0.75 and 1.5 mm BioRad minigels = (1/4); BioRad 1.5 mm supergels = (2); Homemadegels 1.5 mm X 15 cm X 16 cm = (1) 1. The stacking gel is the same as for any gel system: Number of gels

(1/2) (1)

(2)

(3)

(4)

(5)

dH2O

3.7 7.35 14.7 22.05 29.4 36.75

0.5 M Tris-HCl

0.63 1.25 2.5

3.75

5

6.25

10% SDS

0.05 0.1

0.2

0.3

0.4

0.5

30% Acrylamide/0.8% Bis 0.66 1.34 2.68

4.0

5.36

6.7

10% APS

0.05 0.05 0.1

0.15

0.2

0.25

TEMED

0.01 0.01 0.02 0.03 0.04 0.05

Total volume

5

10

20

30

40

2. For a 4-20% gradient gel: 2.1 4% solution: Number of gels

(1/2) (1)

(2)

50

3

dH2O

6

12

50% Glycerol

0.05 0.1

0.2

3 M Tris-HCl, pH 8.8

1.25 2.5

5

10% SDS

0.05 0.1

0.2

30% Acrylamide/0.8% Bis 0.66 1.34 2.68 10% APS

0.05 0.05 0.1

TEMED

0.01 0.01 0.02

Total volume

5

10

20

2.2 20% solution: Number of gels

(1)

(2)

dH2O

0.6

1.2

50% Glycerol

0.1

0.2

3 M Tris-HCl, pH 8.8

2.5

5

10% SDS

0.1

0.2

30% Acrylamide/0.8% Bis

6.6 13.2

10% APS

0.05 0.1

TEMED

0.01 0.02

Total volume

10

20

3. Use a gradient maker, placing the 20% solution in the container right next to the gel and the 4% solution in the container farthest away from the gel. Make sure mixing goes on (the 4% solution volume starts to decrease) as you pour the gradient.

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Polyacrylamide Gel Electrophoresis (PAGE)

POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) Last Update: December 2006 PREPARE SOLUTIONS 1. 30% Acrylamide/ 0.8% Mix 90 g of Acrylamide, 2...

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