Main (12%, 5 Ml Each)Stacking (4 %, 4 Ml Each)

P. Fajerpage 1 of 3

Urea Gels

Hui and Sandy, Ref. Methods in Enzymology 1992, 215,98.

Main (12%, 5 ml each)Stacking (4 %, 4 ml each)

Urea2.4 g2 g

Tris-Gly stock*0.418 ml0.346 ml

Acry (40:1)1.5 ml0.367 ml

d. H2O1 ml1.7 ml

TEMED3 ul3 ul

10% APS30 ul35 ul

Sample Buffer (~0.63 ml)

Urea0.45 g

Tris-Gly stock0.063 ml

d. H2O0.294 ml

DTT (fresh)20 mg

bromophenol blue

Inner Running BufferOuter Running Buffer

Urea36 gTris-Gly

Tris-Gly stock12.5 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.

Example:

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

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

Gels.doc10/20/1999 4:55 PM10/20/1999 4:55 PM