Rehydration Solutions

Some of the chemicals used in the procedures—acrylamide, N,N’-methylenebisacrylamide, ammonium persulfate, TEMED, DTT, iodoacetamide, and DeStreak™ Reagent—are very hazardous. Acrylamide monomer, for example, is a neurotoxin and suspected carcinogen. Read the manufacturer’s safety data sheet (MSDS) detailing the properties and precautions for all chemicals in your laboratory. These safety data sheets should be reviewed prior to starting the procedures described in this handbook. General handling procedures for hazardous chemicals include using double latex gloves for all protocols. Hazardous materials should be weighed in a fume hood while wearing a disposable dust mask. Follow all local rules and regulations for handling and disposal of materials.

A. Sample preparation solution (with urea) for 2-D electrophoresis

[8 M urea, 4% CHAPS, 2% Pharmalyte or IPG buffer (carrier ampholytes), 40 mM DTT, 25 mL]

	Final concentration	Amount
Urea (FW 60.06)	8 M*	12 g
CHAPS†	4% (w/v)	1.0 g
Pharmalyte‡ or IPG Buffer§	2% (v/v)	500 µL
DTT (FW 154.2)	40 mM	154 mg
Double-distilled water	–	to 25 mL (16 mL required)

* If necessary, the concentration of urea can be increased to 9 or 9.8 M.

^† Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octyl glucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).

^‡ Carrier ampholytes (Pharmalyte or IPG buffer) and DTT should be excluded from the sample extraction solution if the samples are to be labeled using 2-D DIGE. See Ettan DIGE User Manual for details.

^§ Use IPG Buffer in the pH range corresponding to the pH range of the IEF separation to be performed, or Pharmalyte in a pH range approximating the pH range of the IEF separation to be performed.

Store in 2.5-mL aliquots at -20 °C.

Note: Protease inhibitors may be added if necessary.

B. Sample preparation solution (with urea and thiourea) for 2-D electrophoresis

[7 M urea, 2 M thiourea, 4% CHAPS, 2% Pharmalyte or IPG Buffer (carrier ampholytes), 40 mM DTT, 25 mL]

	Final concentration	Amount
Urea (FW 60.06)	7 M	10.5 g
Thiourea (FW 76.12)	2 M	3.8 g
CHAPS*	4% (w/v)	1.0 g
Pharmalyte† or IPG Buffer‡	2% (v/v)	500 µL
DTT (FW 154.2)	40 mM	154 mg
Double-distilled water	–	to 25 mL (13.5 mL required)

* Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octyl glucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).

^† Carrier ampholytes (Pharmalyte or IPG buffer) should be excluded from the sample extraction solution if the samples are to be labeled using 2-D DIGE.

^‡ Use IPG Buffer in the pH range corresponding to the pH range of the IEF separation to be performed, or Pharmalyte in a pH range approximating the pH range of the IEF separation to be performed.

Store in 2.5-mL aliquots at -20 °C.

C. Urea rehydration stock solution

(8 M urea, 2% CHAPS, 0.5/2% Pharmalyte or IPG Buffer, 0.002% bromophenol blue, 25 mL)*

	Final concentration	Amount
Urea (FW 60.06)	8 M†	12 g
CHAPS‡	2% (w/v)	0.5 g
Pharmalyte or IPG Buffer§ (same range as the IPG strip)	0.5% (v/v) or 2% (v/v)	125 µL or 500 µL
1% Bromophenol blue stock solution	0.002%	50 µL
Double-distilled water	–	to 25 mL (16 mL required)

* DTT is added just prior to use: 7 mg DTT per 2.5-mL aliquot of rehydration stock solution. For rehydration loading, sample is also added to the aliquot of rehydration solution just prior to use.

^† If necessary, the concentration of urea can be increased to 9 M or 9.8 M.

^‡ Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octyl glucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).

^§ As an alternative to IPG Buffer, use Pharmalyte 3–10 for Immobiline® DryStrip 3–10 or 3–10 NL, Pharmalyte 5–8 for Immobiline® DryStrip 4–7.

^¶ A Pharmalyte/IPG Buffer concentration of 0.5% (125 µL) is recommended with Ettan™ IPGphor 3 Isoelectric Focusing System and an IPG Buffer/Pharmalyte concentration of 2% (500 µL) is recommended with the MultiPhor™ II and Immobiline® DryStrip Kit system.

Store in 2.5-mL aliquots at -20 °C.

D. Thiourea rehydration stock solution

(7 M urea, 2 M thiourea, 2% CHAPS, 0.5/2% Pharmalyte or IPG Buffer, 0.002% bromophenol blue, 25 mL)

	Final concentration	Amount
Urea (FW 60.06)	7 M	10.5 g
Thiourea (FW 76.12)	2 M	3.8 g
CHAPS†	2% (w/v)	1.0 g
Pharmalyte or IPG Buffer	0.5% (v/v) or 2% (v/v)‡	125 µL or 500 µL
1% Bromophenol blue stock solution	0.002%	50 µL
Double-distilled water	–	to 25 mL (13.5 mL required)

* DTT is added just prior to use: Add 7 mg DTT per 2.5-mL aliquot of rehydration stock solution.

^† Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octyl glucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).

^‡ A Pharmalyte/IPG Buffer concentration of 0.5% (125 µL) is recommended with Ettan™ IPGphor 3 Isoelectric Focusing System and an IPG Buffer/Pharmalyte concentration of 2% (500 µL) is recommended with the MultiPhor™ II and Immobiline® DryStrip Kit system.

Store in 2.5-mL aliquots at -20 °C.

E. SDS equilibration buffer solution

(6 M urea, 75 mM Tris-HCl pH 8.8, 29.3% glycerol, 2% SDS, 0.002% bromophenol blue, 200 mL)*

	Final concentration	Amount
Urea (FW 60.06)	6 M	72.1 g
Tris-HCl, pH 8.8 (see solution H)	75 mM	10.0 mL
Glycerol (87% w/w)	29.3% (v/v)	69 mL (84.2 g)
SDS (FW 288.38)	2% (w/v)	4.0 g
1% Bromophenol blue stock solution	0.002%	400 µL
Double-distilled water	–	to 200 mL

* This is a stock solution. Just prior to use, add DTT or iodoacetamide (for first or second equilibration, respectively) as described in the protocol in section 3.1.2.

Store in 20- or 50-ml aliquots at -20 °C.

F. 10× Laemmli SDS electrophoresis buffer

(250 mM Tris base, 1.92 M glycine, 1% SDS, 10 l)*

	Final concentration	Amount
Tris base (FW 121.1)	250 mM	303 g
Glycine (FW 75.07)	1.92 M	1441 g
SDS (FW 288.38)	1% (w/v)	100 g
Double-distilled water	–	to 10 l

* The pH of this solution should not be adjusted.

Store at room temperature.

See also Recipe M for 1× Laemmli SDS electrophoresis buffer.

G. 30% T, 2.6% C monomer stock solution

(30% acrylamide, 0.8% N,N’-methylenebisacrylamide, 1 l)

	Final concentration	Amount
Acrylamide (FW 71.08)	30%	300 g
N,N’-methylenebisacrylamide (FW 154.17)	0.8%	8 g
Double-distilled water	–	to 1 l

Filter solution through a 0.45-µm filter. Store at 4 °C in the dark.

H. 4× resolving gel buffer solution

(1.5 M Tris base, pH 8.8, 1 l)

	Final concentration	Amount
Tris base (FW 121.1)	1.5 M	181.7 g
Double-distilled water	–	750 mL
HClaq	–	adjust to pH 8.8
Double-distilled water	–	to 10 l

Filter solution through a 0.45-µm filter. Store at 4 °C.

I. Bromophenol blue stock solution

	Final concentration	Amount
Bromophenol blue	1%	100 mg
Tris-base	50 mM	60 mg
Double-distilled water	–	to 10 mL

J. 10% SDS solution

(10% SDS, 50 mL)

	Final concentration	Amount
SDS (FW 288.38)	10% (w/v)	5.0 g
Double-distilled water	–	to 50 mL

Filter solution through a 0.45-µM filter. Store at room temperature.

K. 10% ammonium persulfate solution

(10% ammonium persulfate, 10 mL and 1 mL)

	Final concentration	Amount	Amount for 1 mL
Ammonium persulfate (FW 228.20)	10% (w/v)	1.0 g	0.1 g
Double-distilled water	–	to 10 mL	to 1 mL

Fresh ammonium persulfate “crackles” when water is added. If it does not, replace it with fresh stock. Prepare just prior to use.

L. Gel storage solution

(375 mM Tris-HCl, 0.1% SDS, 1 l)

	Final concentration	Amount
4× Resolving gel buffer (see solution H above)	1×	250 mL
10% SDS (see solution J above)	0.1%	10 mL
Double-distilled water	–	to 1 l

Store at 4 °C.

M. 1× Laemmli SDS electrophoresis buffer

(25 mM Tris base, 192 mM glycine, 0.1% SDS, 10 l)*

	Final concentration	Amount
Tris base (FW 121.1)	25 mM	30.3 g
Glycine (FW 75.07)	192 mM	144.0 g
SDS (FW 288.38)	0.1% (w/v)	10.0 g
Double-distilled water	–	to 10 l

* The pH of this solution should not be adjusted.

This solution can be prepared by diluting one volume of 10× Laemmli SDS buffer (solution F) with nine volumes of double-distilled water.

Store at room temperature.

N. Agarose sealing solution

(25 mM Tris base, 192 mM glycine, 0.1% SDS, 0.5% agarose, 0.002% bromophenol blue, 100 mL)

Add all ingredients into a 500-mL Erlenmeyer flask. Swirl to disperse. Heat in a microwave oven on low or on a heating stirrer until the agarose is completely dissolved. Do not allow the solution to boil over. Dispense 1.5-mL aliquots into screw-cap tubes and store at room temperature.