P. Fajer
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Gels
Urea Gel
Ref. Methods in Enzymology 1992, 215,98.
Main (12%, 5 ml each)
Stacking (4 %, 4 ml each)
Urea
2.4 g
2g
Tris-Gly stock*
0.418 ml
0.346 ml
Acry (40:1)
1.5 ml
0.367 ml
d. H2O
1 ml
1.7 ml
TEMED
3 ul
3 ul
10% APS
30 ul
35 ul
Sample Buffer (~0.63 ml)
Urea
0.45 g
Tris-Gly stock
0.063 ml
d. H2O
0.294 ml
DTT (fresh)
20 mg
bromophenol blue
Inner Running Buffer
Outer Running Buffer
Urea
36 g
Tris-Gly
Tris-Gly stock
12.5 ml
d. H2O to
150 ml
Running Condition: 9 mA constant, dye runs off about 60 min. Run extra 60 min.
* Tris-Gly stock (12X): 29.2g Tris + 40g glycine per liter, pH 8.6
Note:
1. All urea-containing buffers are made fresh, since urea spontaneously decomposes to
produce cyanate.
2. Avoid excessive heat with these buffers.
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Example:
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1
2
3
4
5
6
7
8
9
Dephosphorylated LC20
Phosphorylated LC20
LC17
Lane 1,2,6: Dephosphorylated gizzard myosin.
Lane 3-5, 7-9: Phosphorylated gizzard myosin.
Myosin ~ 2 mg/ml, load 10 ul.
Biorad Prep Cell
1. MONOMER SOLUTION (5% GEL WITH NO STACKING)
6.67 mL ACRYL/BIS
23.22 mL H2O
10.0 mL.5 M TIRS/HCL (PH = 8.8)
100 MICRO LITERS APS
10 MICOR LITERS TEMED
2. SAMPLE BUFFER X2
2.5 mL LOW GEL X4
2.0 mL GLYCERIN
.5 mL 20%SDS
100 MICRO LITERS DTT
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page 3 of 4
3. SAMPLE LOADED ONTO GEL
COMBINED 1.5 ML OF 10.5 MG/ML MYO (BA 3/22/95) WITH 1.5 ML OF 10mM
MOPS/1mM EDTA, WITH 3.0 mL OF SAMPLE BUFFER X2
THIS GIVES 15mg OF MYO TOTAL
IN ADDITION TO THIS 40 MICROLITERS OF STANDARD WAS ADDED ONTO
THE GEL. A DESCRIPTION OF PROTEINS IN STANDARD IS ON SECOND PAGE OF ATT
4. RUNNING CONDITIONS OF PREP CELL
INITIAL (1:00 P.M.)
POWER PAC AT 12W CONSTANT WATTAGE
289V AND 42mA
VARIABLE SPEED PUMP AT 55 WHICH YIELDS 100 mL/MIN
GRADIENT MONITOR AT 45(1000MICROS)
PERISTALTIC PUMP 1.0 mL/MIN
UV AT 280 NM
II. RUNNING OF FRACTIONS (PAGE)
12% GEL WITH 2.5% STACKING MADE TO RUN FRACTIONS CORRESPONDING WITH PEAKS
III. RUNNING OF FRACTION II (PAGE)
Silver Staining of SDS gels:
Fixing
>3 h in 40% EtOH/7% AcOH
Wash
2  10 min in 10% EtOH
Wash
3  10 min in Water
Staining
30 min in 0.1% AgNO3
Wash
30 s in Water
Develop
about 15 min in 3% Na2CO3/0.02% Formaldehyde
Stop
10 min in 1% AcOH
Wash
3  10 min in Water
Destain
about 1 min in 0.5% Farmer’s Reagent
(K3[Fe(CN)6]:Na2S2O3·5 H2O, 5:8)
Wash
3  10 min in Water
Intensify
Repeat all steps starting with Staining
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Native Gel Electrophoresis
Native gels are non-denaturing gels.
Proteins are separated on the basis of their charge density (ie. charge to relative mass)
Useful in monitoring formation of protein complexes (eg. Actin:DNase I)
Method
Sample buffer x2
0.1M Tris pH 6.8, 8% glycerol and 0.1% bromophenol blue
(Do not boil samples)
Tank Buffer x10
60g glycine/liter adjusted to pH 8.6 using 2M Tris
Stacking Gel
generally 5% acrylamide
80 mM Tris pH 6.8
Resolving Gel
10-15% acrylamide
x1 Tank buffer
Use TEMED and APS as with SDS-PAGE
Run gels on ice 125V for about 2 Hr. Less time is required if gels are run at RT.
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