Protein Protocols Protein Protocols

188 Ünlü and Minden
4. 4× Stacking gel buffer:
3.0 g of Tris.
Add 40 mL of H 2O.
Adjust pH to 6.8 with 6 N HCl.
Made up to 50 mL with H 2O.
Filter sterilize as described previously and store at –20°C.
5. Light gradient gel solution:
10% acrylamide 8.25 mL of 30% monomer solution
0.375 M Tris 6.25 mL of 4× resolving gel buffer
H 2O 10 mL
0.1% SDS 250 µL of 10% SDS solution
Add these immediately pouring the gel:
APS 82.5 µL of 10% stock solution
TEMED 8.25 µL
6. Heavy gradient gel solution:
15% Acrylamide 12.25 mL of 30% monomer solution
0.375 M Tris 6.25 mL of 4× resolving gel buffer
H 2O 3.8 mL
0.1% SDS 250 µL of 10% SDS solution
Sucrose 3.75 g
Add these immediately before pouring the gel:
Ammonium pensulfate (APS)82.5 µL of 10% stock solution
N,N,N',N'-tetramethylethylenediamine (TEMED) 8.25 µL
7. Stacking gel solution:
3.5 % Acrylamide 400 µL of 30% monomer solution
0.175 M Tris 800 µL of 4× stacking gel solution
H 2O 2.0 mL
0.1% SDS 33 µL of 10% SDS solution
Add these two just before pouring the gel:
APS 16.7 µL of 10% stock solution
TEMED 1.7 µL
2.4. Image Acquisition and Analysis
1. The cooled CCD camera is a 16-bit, series 300 model purchased from Photometrics/Roper
Scientific (Tucson, AZ). It is fitted with a standard 105 mm macro lens from Nikon,
available from most photographical suppliers. Two 250 W quartz–tungsten–halogen lamps
from Oriel (Stratford, CT) are used for illumination. Single-bandpass excitation filters
2.5 cm diameter from Chroma Technology (Brattleboro, VT), are used to excite 545 ±
10 nm and 635 ± 15 nm for Cy3 and Cy5, respectively. A multiwavelength bandpass
emission filter from Chroma Technology is used to image the gels at 587.5 ± 17.5 nm and
695 ± 30 nm for Cy3 and Cy5, respectively. The imager housing was constructed in-house
from black Plexiglas. Image acquisition is semiautomated and is controlled by an Silicon
Graphics. (Mountain View, CA) O2 computer workstation.
2. Destain solution:
1% Acetic acid 10 mL of glacial acetic acid
40% Methanol 400 mL
H2O 590 mL