8.5 M Urea Stock Solution
Final Volume
1 Liter
500 ml
250 ml
10 ml
Urea
510 g
255 g
127.5 g
5.1 g
Initial Water
600 ml
300 ml
150 ml
6 ml
Dissolve the urea in nanopure water with gentle heating (<30°C) and vigorous stirring with a heavy stirbar.
Remove from the heat source as soon as the urea dissolves. Aliquot into 41.1 ml and store -80 ºC. Thaw immediately before use and discard any remaining solution. Do not leave solution at RT or 4 ºC any longer than necessary.
Standard Rehydration Solution
Components Amount for 5 ml Amount for 10 ml Amount for 50 ml Final
Concentration
8.5 M Urea
Thiourea
CHAPS
40% Ampholytes 3-10
0.2% Bromophenol Blue*
Nanopure water
DTT
4.11 ml
0.761 g
0.2 g
63 μl
5 μl
Adjust to 5 ml
0.004 g / ml
8.22 ml
1.52 g
0.4 g
125
μl
10 μl
Adjust to 10 ml
0.004 g / ml
41.1 ml
7.61 g
2 g
625 μl
50 μl
Adjust to 50 ml
0.004 g / ml
7 M
2 M
4%
0.5%
0.0002%
------
0.4% ≈ 26 mM
* Optional component.
Add Thiourea, CHAPS, Ampholytes, and Bromophenol Blue to Urea solution. Aliquot into 0.5 & 1 ml aliquots and then immediately store at -80ºC. Thaw only the required number of aliquots and discard leftover solution.
Add DTT immediately before use.
Note: Thiourea is classified by OHSA as a “SELECT CARCINOGEN” and so special precautions and training must be taken when using this reagent.
UTC Rehydration Solution (DIGE)
Components
8.5 M Urea
Thiourea
CHAPS
Nanopure water
Amount for 10 ml
8.22 ml
1.52 g
0.4 g
Adjust to 5 ml
Amount for 50 ml
41.1 ml
7.61 g
2 g
Adjust to 50 ml
Final Concentration
7 M
2 M
4%
------
Add Thiourea & CHAPS to Urea solution. Aliquot into 1 ml aliquots and then immediately store at -80ºC.

ASB-14 Spiking
For better resolution of membrane and membrane associated proteins, add 0.4% ASB-14. ASB-14 interferes with DIGE labeling so spike it after labeling.
1.5 M Tris, pH 8.8
Components
Tris
Nanopure water
Amount for 100 ml
18.17 g
Adjust to 100 ml
Filter sterilize to stop from growing or freeze.
Equilibration Base Buffer
Components Amount for 100 ml
Urea
20% SDS
1.5 M Tris-HCl, pH 8.8
100% Glycerol
Nanopure water
36 g
10 ml
3.3 ml
20 ml
Adjust to 100 ml
Amount for 500 ml
180 g
50 ml
16.5 ml
100 ml
Adjust to 500 ml
Final Concentration
7 M
2%
0.05 M
20 %
------
Add Urea, SDS, Tris Buffer and Glycerol to half the final volume of nanopure water. Don’t heat to get into solution. Aliquot into 8 & 40 ml aliquots and then immediately store at -80ºC. To make DTT Equilibration
Buffer 1, add 200 mg DTT to 10 ml of Base Buffer [2%] immediately prior to use. To make Iodoacetamide
Equilibration Buffer 2, add 250 mg iodoacetamide to 10 ml of Base Buffer [2.5%] immediately prior to use.
Agarose Overlay Solution
Components Amount for 50 ml Final Concentration
Agarose (low melt)
10X SDS-PAGE Running Buffer
0.2% Bromophenol Blue
Nanopure water
0.25 g
5 ml
0.2 ml
45 ml
0.5%
1X
0.008%
------
Make the solution in a 100 ml plastic polypropylene Nalgene-like bottle. Melt the agarose in the microwave.
Stop after 10 sec and swirl bottle to mix. Then continue microwaving for 5 additional sec and swirling until the agarose goes into solution. Since the solution has SDS, the solution will boil over very quickly. Store the agarose overlay solution at 4°C after it has completely cooled on the bench top. If you put the agarose solution in the refrigerator before it has completely cooled, the bottle will collapse.

10X SDS-PAGE Running Buffer
Components
Tris
Glycine
SDS
Nanopure water
Amount for 1 L
30.3 g
144.1 g
10 g
Adjust to 1 L
Amount for 3 L Amount for 20 L
90.8 g
432.3 g
30 g
Adjust to 3 L
605 g
2882 g
200 g
Adjust to 20 L
Final
Concentration
250 mM, pH 8.3
1920 mM
1%
------

Gel Storage Solution
Make 1 M (6.5%) sodium azide stock = 3.25g per 50 ml nanopure water.
Dilute stock to 0.005% solution
(769 μl 6.5% stock per 1000 ml water) in autoclaved nanopure water and then add to gels. Store gels at 4ºC.
Chemical Suppliers
Reagent Catalog number Vender
2007 UCLA
Prices
Bio-Lyte 3/10 Ampholytes
CHAPS
DTT (Cleland’s Reagent)
Glycerol (OmiPur 99%)
NP-40 Alternative
Thiourea
Urea (Omipur 99.5%)
Prepared by Melissa Sondej version 6/8/11.
163-1112
PI28300
233155
4750
492016
T-8656
9530
Bio-Rad
Fisher
Calbiochem/VWR
EMD/VWR
Calbiochem/VWR
Sigma
EMD/VWR
$100.70