Ettan DALTsix Electrophoresis Unit, 115 VAC

Quick guide for finding information on gel casting for DALTsix and DALTtwelve systems

To find gel-casting information quickly, refer to Table 2 for gel volumes required, Table 3 for single percentage gel recipes, and Table 4 for gradient gel recipes.

Warning: Some of the chemicals used in the procedures that follow—acrylamide, N,N’-methylenebisacrylamide, ammonium persulfate, TEMED, thiourea, DTT, and iodoacetamide—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 safety rules and regulations, including for disposal.

Table 2. Volumes required per cast (Ettan DALT systems).

Casting System	Volume (ml)
Ettan DALTsix
6 gels x 1-mm thick spacers	450
6 gels x 1.5-mm thick spacers	600
Ettan DALTtwelve
14 gels x 1-mm thick spacers	90
13 gels x 1.5-mm thick spacers	1200

Table 3a. Single-percentage gel recipes for Ettan DALT systems.*

Final gel concentration	10%	12.5%	15%
Monomer solution	300 ml	375 ml	450 ml
4x resolving gel buffer	225 ml	225 ml	225 ml
10% SDS	9 ml	9 ml	9 ml
Double-distilled water	360.7 ml	285.7 ml	210.8 ml
10% ammonium persulfate †	5 ml	5 ml	5 ml
TEMED †	0.30 ml	0.25 ml	0.20 ml
Total volume	900 ml	900 ml	900 ml

* Preparation of stock solutions is described in the end of this chapter. Adjust as necessary for the thickness of the gels and the number of gels cast.
† Add after (optional) deaeration.

Table 3b. Single-percentage gel recipes for Ettan DALT systems using premade solution of acrylamide and bisacrylamide.* 

Final gel concentration	10%	12.5%	15%
PlusOne ReadySol IEF 40%T, 3%C	225 ml	281.25 ml	337.5 ml
4x resolving gel buffer	225 ml	225 ml	225 ml
10% SDS	9 ml	9 ml	9 ml
Double-distilled water	436.2 ml	380 ml	323.8 ml
10% ammonium persulfate †	4.5 ml	4.5 ml	4.5 ml
TEMED †	0.30 ml	0.25 ml	0.20 ml
Total volume	900 ml	900 ml	900 ml

* Preparation of stock solutions is described in the end of this chapter. Adjust as necessary for the thickness of the gels and the number of gels cast.

Table 4a. Recipes for gradient gels for Ettan DALT systems.*

Light solution - Final concentration	8%	10%	12%	14%	16%
Monomer solution	120 ml	150 ml	180 ml	210 ml	240 ml
4x resolving gel buffer	113 ml	113 ml	113 ml	113 ml	113 ml
10% SDS	4.5 ml	4.5 ml	4.5 ml	4.5 ml	4.5 ml
Double-distilled water	210.5 ml	180.5 ml	150.5 ml	120.5 ml	90.5 ml
10% ammonium persulfate †	1.8 ml	1.8 ml	1.8 ml	1.8 ml	1.8 ml
TEMED †	0.225 ml	0.225 ml	0.225 ml	0.225 ml	0.225 ml
Total volume	450 ml	450 ml	450 ml	450 ml	450 ml

Heavy solution - Final concentration	12%	14%	16%	18%	20%
Monomer solution	180 ml	210 ml	240 ml	270 ml	300 ml
4x resolving gel buffer	113 ml	113 ml	113 ml	113 ml	113 ml
Glycerol (87% [w/w])	31 ml	31 ml	31 ml	31 ml	31 ml
10% SDS	4.5 ml	4.5 ml	4.5 ml	4.5 ml	4.5 ml
Double-distilled water	119.9 ml	89.9 ml	59.9 ml	29.9 ml	0 ml
10% ammonium persulfate †	1.4 ml	1.4 ml	1.4 ml	1.4 ml	1.4 ml
TEMED †	0.225 ml	0.225 ml	0.225 ml	0.225 ml	0.225 ml
Total volume	450 ml	450 ml	450 ml	450 ml	450 ml

* Preparation of stock solutions is described in the end of this chapter. Adjust as necessary for the thickness of the gels and the number of gels cast.
† Add after (optional) deaeration.

Table 4b. Recipes for gradient gels for Ettan DALT systems using premade solution of acrylamide and bisacrylamide.*

Light solution - Final concentration	8%	10%	12%	14%	16%
PlusOne ReadySol IEF 40%T, 3%C	90 ml	112.5 ml	135 ml	157.5 ml	180 ml
4x resolving gel buffer	113 ml	113 ml	113 ml	113 ml	113 ml
10% SDS	4.5 ml	4.5 ml	4.5 ml	4.5 ml	4.5 ml
Double-distilled water	240.5 ml	218 ml	195.5 ml	173 ml	150.5 ml
10% ammonium persulfate †	1.8 ml	1.8 ml	1.8 ml	1.8 ml	1.8 ml
TEMED †	0.225 ml	0.225 ml	0.225 ml	0.225 ml	0.225 ml
Total volume	450 ml	450 ml	450 ml	450 ml	450 ml

Heavy solution - Final concentration	12%	14%	16%	18%	20%
PlusOne ReadySol IEF 40%T, 3%C	135 ml	157.5 ml	180 ml	202.5 ml	225 ml
4x resolving gel buffer	113 ml	113 ml	113 ml	113 ml	113 ml
Glycerol (87% [w/w])	31 ml	31 ml	31 ml	31 ml	31 ml
10% SDS	4.5 ml	4.5 ml	4.5 ml	4.5 ml	4.5 ml
Double-distilled water	164.9 ml	142.4 ml	119.9 ml	97.4 ml	74.9 ml
10% ammonium persulfate †	1.4 ml	1.4 ml	1.4 ml	1.4 ml	1.4 ml
TEMED †	0.225 ml	0.225 ml	0.225 ml	0.225 ml	0.225 ml
Total volume	450 ml	450 ml	450 ml	450 ml	450 ml

* Preparation of stock solutions is described in the end of this chapter. Adjust as necessary for the thickness of the gels and the number of gels cast.

30 % T, 2.6 % C monomer stock solution
(30% acrylamide, 0.8% N,N’-methylenebisacrylamide, 1 l)

	Final concentration	Amount
Acrylamide (PF 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.

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 1 l

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

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-HCI, pH 8.8**	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% (w/v)	400 µ
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) 
** For preparing the Tris-HCl, pH 8.8 solution see Table 3.
Store in 20- or 50-ml aliquots at -20 °C.

10% ammonium persulfate solution
(10% ammonium persulfate, 10 ml and 1 ml)

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

Preparing the gel caster for Ettan DALTsix system Set up the gel caster near a sink, in a tray, or on a drain board so that any liquid that overflows, spills, or drains out of the unit during pouring or disassembly can be easily contained. The Ettan DALTsix Gel Caster accommodates up to six 1-mm or 1.5-mm gel cassettes with separator sheets (0.5 mm) between them. If you are not planning to cast a full set of gels, use the blank cassette inserts (purchased separately) with separator sheets between them to occupy the extra space. Gel labels, for easy indexing of gels and samples, can be placed in the cassettes at any time during the assembly of the caster. 1. Check that the caster is level. Remove the faceplate and lay the caster on its back. If casting gels by the displacement method, remove the triangular wedge in the V-shaped base. 2. Fill the gel caster starting with a separator sheet against the back wall. The separator sheets make it easier to remove the cassettes from the unit after polymerization. Fill the caster by alternating cassettes with separator sheets. The rubber hinge should be on the left side of the caster, with the matching ends of the cassette down. End with a separator sheet then use the thicker filler sheets (1.0 mm) to bring the level of the stack of cassettes and spacers even with the edge of the caster. 3. Remove the gray foam seal from the groove in the faceplate and lubricate it with a light coating of GelSeal compound to help ensure a liquid-tight seal. Place the gasket back in the groove on the faceplate. Avoid stretching the gasket by seating it from the ends first, working toward the middle. 4. Turn two black knobbed screws into the two threaded holes across the bottom until they are well engaged (two to three full turns). Carefully place the faceplate onto the caster with the bottom slots resting on their respective screws. Clamp both sides of the faceplate with six spring clips and tighten the screws. Be sure the sealing gasket is compressed evenly by the faceplate and forms a tight seal with the caster. Do not overtighten the screws. 5. If casting using the filling channel in the back plate of the caster, be sure to plug the barbed fitting in the faceplate. If casting using the displacement method, attach the tubing from the peristaltic pump to the barbed fitting. Fig 1. Gel caster for DALTsix system.

Gel casting for DALTsix and DALTtwelve systems

The instructions provided below for the preparation of vertical SDS-polyacrylamide gels employ the Tris-glycine system of Laemmli. Vertical second-dimension gels are most conveniently cast several at a time, in a multiple gel caster.

Warning: Some of the chemicals used in the procedures that follow—acrylamide, N,N’-methylenebisacrylamide, ammonium persulfate, TEMED, thiourea, DTT, and iodoacetamide—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 safety rules and regulations, including for disposal.

1. Select the gel percentage
a. Single percentage gel versus gradient gel. When a gradient gel is used, the overall separation interval is wider and the linear separation interval is larger. In addition, sharper bands result because the decreasing pore size functions to minimize diffusion. However, a gradient gel requires more skill to cast. For detailed instructions on gradient preparation, see the user manual for the relevant electrophoresis unit and multiple gel caster.

Single percentage gels offer better resolution for a particular M_r window. A commonly used second-dimension gel for 2-D electrophoresis is a homogeneous gel containing 12.5% total acrylamide.

Note: Stacking gels are not necessary for vertical 2-D gels.

b. Whether single percentage or gradient, the appropriate percentage gel is selected according to the range of separation desired (Table 1).

Table 1. Recommended acrylamide concentrations for protein separation.

Acrylamide percentage in resolving gel		Separation size range (Mr x 10-3)
Single percentage	5	36 - 200
	7.5	24 - 200
	10	14 - 200
	12.5	14 - 100*
	15	14 - 60*
Gradient	5 - 15	14 - 200
	5 - 20	10 - 200
	10 - 20	10 - 150

* Larger proteins fail to move significantly into the gel.

2. Select gel thickness and calculate casting solution volume
DALT gel casting cassettes with either 1.0- or 1.5-mm-thick spacers can be used. Thinner gels stain and destain more quickly and generally give less background staining. Thicker gels have a higher protein capacity. Thicker gels are also less fragile and easier to handle.
Table 2 gives the volumes required for Ettan DALT systems.

Table 2. Volumes required per cast (Ettan DALT systems).

Casting System	Volume (ml)
Ettan DALTsix
6 gels x 1-mm thick spacers	450
6 gels x 1.5-mm thick spacers	600
Ettan DALTtwelve
14 gels x 1-mm thick spacers	900
13 gels x 1.5-mm thick spacers	1200

3. Calculate the formulation of the gel solution
The recipes given in Table 3a and 3b (premade solution of acrylamide and bisacrylamide) produce 900 ml of solution for a single percentage gel. The recipes in Table 4a and 4b (premade solution of acrylamide and bisacrylamide) produce 450 ml each of light and heavy solution for a gradient gel. These recipes can be scaled up or down, depending on the volume required.

Table 3a. Single-percentage gel recipes for Ettan DALT systems.*

Final gel concentration	10%	12.5%	15%
Monomer solution	300 ml	375 ml	450 ml
4x resolving gel buffer	225 ml	225 ml	225 ml
10% SDS	9 ml	9 ml	9 ml
Double-distilled water	360.7 ml	285.7 ml	210.8 ml
10% ammonium persulfate †	5 ml	5 ml	5 ml
TEMED †	0.30 ml	0.25 ml	0.20 ml
Total volume	900 ml	900 ml	900 ml

* Preparation of stock solutions is described in the end of this chapter. Adjust as necessary for the thickness of the gels and the number of gels cast.
† Add after (optional) deaeration.

Table 3b. Single-percentage gel recipes for Ettan DALT systems using premade solution of acrylamide and bisacrylamide.* 

Final gel concentration	10%	12.5%	15%
PlusOne ReadySol IEF 40%T, 3%C	225 ml	281.25 ml	337.5 ml
4x resolving gel buffer	225 ml	225 ml	225 ml
10% SDS	9 ml	9 ml	9 ml
Double-distilled water	436.2 ml	380 ml	323.8 ml
10% ammonium persulfate †	4.5 ml	4.5 ml	4.5 ml
TEMED †	0.30 ml	0.25 ml	0.20 ml
Total volume	900 ml	900 ml	900 ml

* Preparation of stock solutions is described in the end of this chapter. Adjust as necessary for the thickness of the gels and the number of gels cast.
† Add after (optional) deaeration.

Table 4a. Recipes for gradient gels for Ettan DALT systems.*

Light solution - Final concentration	8%	10%	12%	14%	16%
Monomer solution	120 ml	150 ml	180 ml	210 ml	240 ml
4x resolving gel buffer	113 ml	113 ml	113 ml	113 ml	113 ml
10% SDS	4.5 ml	4.5 ml	4.5 ml	4.5 ml	4.5 ml
Double-distilled water	210.5 ml	180.5 ml	150.5 ml	120.5 ml	90.5 ml
10% ammonium persulfate †	1.8 ml	1.8 ml	1.8 ml	1.8 ml	1.8 ml
TEMED †	0.225 ml	0.225 ml	0.225 ml	0.225 ml	0.225 ml
Total volume	450 ml	450 ml	450 ml	450 ml	450 ml

Heavy solution - Final concentration	12%	14%	16%	18%	20%
Monomer solution	180 ml	210 ml	240 ml	270 ml	300 ml
4x resolving gel buffer	113 ml	113 ml	113 ml	113 ml	113 ml
Glycerol (87% [w/w])	31 ml	31 ml	31 ml	31 ml	31 ml
10% SDS	4.5 ml	4.5 ml	4.5 ml	4.5 ml	4.5 ml
Double-distilled water	119.9 ml	89.9 ml	59.9 ml	29.9 ml	0 ml
10% ammonium persulfate †	1.4 ml	1.4 ml	1.4 ml	1.4 ml	1.4 ml
TEMED †	0.225 ml	0.225 ml	0.225 ml	0.225 ml	0.225 ml
Total volume	450 ml	450 ml	450 ml	450 ml	450 ml

* Preparation of stock solutions is described in the end of this chapter. Adjust as necessary for the thickness of the gels and the number of gels cast.
† Add after (optional) deaeration.

Table 4b. Recipes for gradient gels for Ettan DALT systems using premade solution of acrylamide and bisacrylamide.*

Light solution - Final concentration	8%	10%	12%	14%	16%
PlusOne ReadySol IEF 40%T, 3%C	90 ml	112.5 ml	135 ml	157.5 ml	180 ml
4x resolving gel buffer	113 ml	113 ml	113 ml	113 ml	113 ml
10% SDS	4.5 ml	4.5 ml	4.5 ml	4.5 ml	4.5 ml
Double-distilled water	240.5 ml	218 ml	195.5 ml	173 ml	150.5 ml
10% ammonium persulfate †	1.8 ml	1.8 ml	1.8 ml	1.8 ml	1.8 ml
TEMED †	0.225 ml	0.225 ml	0.225 ml	0.225 ml	0.225 ml
Total volume	450 ml	450 ml	450 ml	450 ml	450 ml

Heavy solution - Final concentration	12%	14%	16%	18%	20%
PlusOne ReadySol IEF 40%T, 3%C	135 ml	157.5 ml	180 ml	202.5 ml	225 ml
4x resolving gel buffer	113 ml	113 ml	113 ml	113 ml	113 ml
Glycerol (87% [w/w])	31 ml	31 ml	31 ml	31 ml	31 ml
10% SDS	4.5 ml	4.5 ml	4.5 ml	4.5 ml	4.5 ml
Double-distilled water	164.9 ml	142.4 ml	119.9 ml	97.4 ml	74.9 ml
10% ammonium persulfate †	1.4 ml	1.4 ml	1.4 ml	1.4 ml	1.4 ml
TEMED †	0.225 ml	0.225 ml	0.225 ml	0.225 ml	0.225 ml
Total volume	450 ml	450 ml	450 ml	450 ml	450 ml

4. Prepare the gel solution
Make up the gel solution without TEMED or ammonium persulfate.

Note: An optional deaeration step may be performed at this point. To do so, make up the solution in a vacuum flask. Add a small magnetic stirring bar. Stopper the flask and apply a vacuum for several minutes while stirring on a magnetic stirrer.

Just before casting the gel, add TEMED and 10% ammonium persulfate. Gently swirl the flask to mix, being careful not to generate bubbles. Immediately pour the gel.

5. Pour and prepare the gel
Fill the gel cassette to 5–10 mm below the top (no stacking gel layer is required).

Overlay each gel with a layer of water-saturated 1-butanol (1.0 ml) immediately after pouring to minimize gel exposure to oxygen and to create a flat gel surface.

After allowing a minimum of 2 h for polymerization, remove the overlay and rinse the gel surface with gel storage solution (Table 5).

Table 5. Gel storage solution.
(375 mM Tris-HCl, 0.1% SDS, 1 l)

	Final concentration	Amount
4x resolving gel buffer	1×	250 ml
10% SDS	0.1%	10 ml
Double-distilled water	-	to 1 l

Store at 4 °C.

Note: An alternative to using water-saturated 1-butanol to overlay the gels after casting is to spray the edges of the cassettes using a 0.1% (w/v) SDS/water solution (using a plant sprayer) such that the edges are covered by just a few millimeters. This technique helps to avoid curved edges on the gels.

6. Storage of unused gels
Gels not used immediately can be stored for future use at 4 °C for up to two weeks. Gel storage solution (see Table 5) is pipetted over the top gel surface and the gel cassette is sealed with flexible paraffin film.

Alternatively, the gel cassettes can be stored fully immersed in gel storage solution.
For further information on the preparation of second-dimension vertical SDS slab gels, refer to the user manuals for the respective vertical gel unit and multiple gel caster.

30 % T, 2.6 % C monomer stock solution
(30% acrylamide, 0.8% N,N’-methylenebisacrylamide, 1 l)

	Final concentration	Amount
Acrylamide (PF 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.

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 1 l

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

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-HCI, pH 8.8**	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% (w/v)	400 µ
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) 
** For preparing the Tris-HCl, pH 8.8 solution see Table 3.
Store in 20- or 50-ml aliquots at -20 °C.

10% ammonium persulfate solution
(10% ammonium persulfate, 10 ml and 1 ml)

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

For convenient run times and minimal spot diffusion we recommend the following run conditions.

Table 4. Day Run — Temperature 22°C.

Step	mA/gel	Voltage (V)	W/gel	Time (hours)
A	10	80	1	1
B	50	500	171	4 - 5

¹. Maximum electrical input for the electrophoresis unit is 600 V, 400 mA, and 100 W.

Table 5. Overnight Run — Temperature 15°C.

Step	mA/gel	Voltage (V)	W/gel	Time (hours)
A	12	150	1.5	15 - 17*

^* Change to day run protocol when ever convenient.

Stop run
Continue the electrophoresis until the bromophenol blue front reaches the end of the gel. The front can be run off the gel if desired.

Removing the gels
1. Remove the lid and carefully lift the UBC upwards making sure the cassettes remain in the anode assembly by applying downward pressure on them.
2. Lift the gel cassette holder upwards and remove the gels.

Care and Maintenance 
1. On completion of the electrophoresis run, immediately remove the UBC from the Ettan DALTsix Electrophoresis Unit.
2. Wash the UBC in distilled water of room temperature to wash off the buffer solution. Rinse thoroughly and allow the UBC to dry at room temperature.
3. Do not leave the UBC in buffer solution when the unit is not in use. Store the UBC empty, not even on the cassette holder, since the sealings in the middle.

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 mM	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.

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 with nine volumes of double-distilled water.
Store at room temperature.

2× Laemmli SDS electrophoresis buffer
(50 mM Tris base, 384 mM glycine, 0.2% SDS, 10 l)*

	Final concentration	Amount
Tris base (FW 121.1)	50 mM	60.6 g
Glycine (FW 75.07)	384 mM	288.0 g
SDS (FW 288.38)	0.2% (w/v)	20.0 g
Double-distilled water	-	to 10 l

* The pH of this solution should not be adjusted.
This solution can be prepared by diluting two volumes of 10× Laemmli SDS buffer with eight volumes of double-distilled water.
Store at room temperature.

1. Open the gel package
Cut around the package on two sides at approximately 1 cm from the edge to avoid cutting the gel or the support film. Remove the gel from the package. The gel is cast onto a plastic support film and does not cover the film entirely. The gel is covered with a protective plastic sheet. Markings on the protective sheet indicate the orientation of the gel and the direction of electrophoresis. The bottom (+ or anodic) edge of the gel is flush with the edge of the support film. The support film protrudes approximately 15 mm beyond the top (- or cathodic) edge of the gel and approximately 5 mm at either side.

2. Open DALT Precast Gel Cassette
Place the gel cassette on the bench top with the hinge down. Apply 1 ml gel buffer onto the glass plate as a streak along the spacer on the right edge of the glass plate (Fig 2). Add an additional 2 ml of gel buffer to the center of the plate.


Fig 2. Pipetting a streak of gel buffer onto the glass plate. The arrow indicates the direction of motion in applying the streak.

3. Remove the protective plastic sheet from the gel
Handling the gel only by the side support film margins, hold it (gel-side down) over the glass plate. Ensure that it is oriented with the cathodic (-) edge of the gel toward the cathodic (-) edge of the cassette. Align the right edge of the gel with the right edge of the side spacer of the glass plate side, flex the gel downward slightly and lower it slowly toward the glass plate from right to left. Take care that the bottom (anodic) edge of the gel is flush (within 1 mm) of the bottom (anodic) edge of the glass plate. The protruding side support film margins (but not the gel) should rest on top of the side spacers.

4. Remove bubbles and excess buffer
Use the roller (a separate accessory) to press out any bubbles or liquid from between the gel and the glass. Press firmly against the plastic support film with the roller and roll over the entire gel (Fig 3). After rolling, the gel should adhere firmly to the glass and resist further movement.


Fig 3. Pressing out air pockets between gel and glass plate.

5. Close the cassette
Close the cassette, snap the plastic frame to the glass plate (Fig 4) and press the edges tightly together along the entire side of the cassette. Ensure that the cassette is closed completely; an incompletely closed cassette causes a strongly curved front.


Fig 4. Closing the DALT Precast Gel Cassette.


6. Repeat the procedure for each second-dimension gel to be run

The second-dimension vertical gel must be ready for use prior to Immobiline DryStrip gel equilibration.

Equilibration is carried out in a two-step process using tubes and volume of equilibration solution as specified in the table 1 below.

Preparatory steps
1. Place the IPG strips in individual tubes, with the support film toward the tube wall.

2. Prepare an appropriate volume of SDS equilibration buffer solution (see Table 2 below) then measure into two equal volumes. Add DTT to one portion (100 mg per 10 ml) and iodoacetamide to the other (250 mg per 10 ml).

Equilibration
1. Add the appropriate volume of SDS equilibration buffer (+ DTT) to each strip. Cap or seal the tubes with flexible paraffin film and place them on their sides on a rocker for the equilibration process. Equilibrate for 15 min.

2. Pour off buffer from above step and add the appropriate volume of SDS equilibration buffer (+iodoacetamide) to each strip. Again cap or seal the tubes with flexible paraffin film and place them on their sides on a rocker for the equilibration process. Equilibrate for an additional 15 min.

Note: Be consistent with the timing of the equilibration steps.

Note: The subsequent steps of gel assembly, preparation of electrophoresis unit, insertion of the gel into the precast gel cassette, and melting of the sealing solution can be performed as the Immobiline DryStrip gels are equilibrating, as long as the timeframes above are adhered to.

Table 1. Suggested containers and volumes of equilibration solution.

Strip length (cm)	Container	Equilibration solution (ml)
7	Disposable, 15-ml conical tubes	2.5 - 5
11	25 × 200 mm screw-cap culture tubes	5 - 10
13	25 × 200 mm screw-cap culture tubes	5 - 10
18	25 × 200 mm screw-cap culture tubes, Equilibration tubes or Petri dish	10 - 15
24	Equilibration tubes or Petri dish	10 - 15

Table 2. 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**	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% (w/v)	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).
** For preparing the Tris-HCl, pH 8.8 solution see Table 3.

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

Table 3. 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
HCI	-	adjust to pH 8.8
Double-distilled water	-	to 1 l

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

1. Carefully insert the electrophoresis gels (Fig 5) and fill any empty slots with DALT blank cassette inserts.


Fig 5. Inserting the cassettes into the cassette carrier.


2. Sealing and filling the Upper buffer chamber.

Wet the Upper buffer chamber (UBC) sealings with buffer solution or 0.1% SDS (immerse the sealings in solution or spray the sealings of the UBC using a plant sprayer) and carefully slide the UBC over the gel cassettes. Do not move the UBC repeatedly up and down as this will reduce the sealing effect. Fill the UBC with 1.2 liters of 2x Laemmli SDS electrophoresis buffer, refer to section 6, Buffer recipes. and use a funnel to adjust the buffer level in the lower buffer chamber to the same height as in the UBC, by adding 1x Laemmli SDS electrophoresis buffer, refer to section 6, Buffer recipes.


3. Hydrostatic balance.

By adjusting the buffer level in the Lower buffer chamber to be the same as the level in the UBC, you achieve a status called ‘hydrostatic balance’, where there will be no overall change in the levels of either buffer chamber during electrophoresis. This will also help to improve runs and minimize any possible ‘leakage’ from the UBC. See Fig 6.


Fig 6. Adjusting the buffer level.

1. When preparing to run a gel in the DALTsix instrument, insert the anode assembly in the tank and then fill the lower buffer chamber tank. Add one bottle (125 ml) of DIGE Anode Buffer stock solution included in the DIGE Buffer Kit into the tank. Fill the electrophoresis unit to the 4.5 l fill line with distilled or deionized water, and turn the pump on.
Note: Use only one bottle of the DIGE Anode (lower) Buffer for each DALTsix run.

2. Turn on the circulation pump by plug the leads attached to DALTsix base unit. (Only perform this operation once the tank contains buffer.)

3. Connect an external thermostatic circulator (e.g. MultiTemp III) and set the temperature to 22ºC for a day run and to 15ºC for an overnight run. Equilibrate the buffer to 15ºC before starting an overnight run.

Note: Place the unit close to a sink for easy rinsing and draining. Connect the tubing leading to and from the heat exchanger to a thermostatic temperature controller such as MultiTemp III. Do not connect the heat exchanger to a water tap or any other coolant supply that does not have pressure regulation. Position a suitable power supply (e.g. EPS 601) conveniently close to the electrophoresis unit.

1. Prepare anode and cathode buffers (stocks included in the DALT Buffer Kit)
Dilute half of the 100× anode (lower) buffer by adding 37.5 ml to 4.5 l of water.
Dilute one bottle of 10× cathode (upper) buffer to a final volume of 0.8 l with deionized water.

2. Prepare anode assembly
When preparing to run a gel in the Ettan DALTsix Electrophoresis Unit, insert the anode assembly in the tank and then fill the lower buffer chamber tank with 4.5 liters of 1x electrophoresis buffer (Fig 1.) Turn on the circulation pump by plug the leads attached to the DALTsix base unit. (Only perform this operation once the tank contains buffer.)


Fig 1. Filling the Ettan DALTsix electrophoresis unit with anode buffer.

3. Temperature control
Connect an external thermostatic circulator (e.g. MultiTemp™ III) and set the temperature to 25°C for a day run and to 30°C for an overnight run.

The equilibration step saturates the Immobiline™ DryStrip gel (IPG strip) with the SDS buffer system required for the second-dimension separation. To reduce vertical streaking in the second dimension it is necessary to apply two equilibration steps. The first step saturates the IPG strip with the SDS system and the second step blocks the protein thiol groups. The equilibration solution contains buffer, urea, glycerol, reductant, SDS and dye.

Prepare equilibration solution. Prepare SDS equilibration buffer (Table 1). This is a stock solution. Just prior to use, add 50 mg DTT per 10 ml SDS equilibration buffer (0.5% [w/v]).

Table 1. SDS equilibration buffer.

	Final concentration	Amount
1.5 M Tris-Cl, pH 8.8	50 mM	6.7 ml
Urea (FW 60.06)	6 M	72.07 g
Glycerol (87% w/w)	30% (v/v)	69 ml
SDS (FW 288.38)	2% (w/v)	4.0 g
Bromophenol blue	0.001% (w/v)	2 mg
Distilled or deionized water	-	to 200 ml

Store in 40 ml aliquots at –20ºC.

Equilibration. 
Place the IPG strips in individual tubes with the support film toward the tube wall. Add 10 ml DTT-containing solution to each tube. Place the tubes on a rocker and equilibrate for 15 min.
Note: When using CyDye DIGE saturation dyes (Labeling Kit for scarce samples), repeat the first equilibration with DTT-containing SDS equilibration solution for another 15 min.

Second equilibration. 
A second equilibration is performed with an iodoacetamide solution (instead of DTT). Prepare a solution of 450 mg iodoacetamide per 10 ml of SDS equilibration buffer (4.5% [w/v]). Decant the first equilibration solution and add the same volume of iodoacetamide containing equilibration solution to each tube. Place the tubes on a rocker and equilibrate for an additional 15 min.

Note: The subsequent steps of electrophoresis unit preparation, insertion of gels and melting of the Sealing Solution can be performed while the IPG strips are equilibrating

1. Carefully insert the electrophoresis gels (Fig 4) and fill any empty slots with DALT blank cassette inserts.


Fig 4. Inserting the cassettes into the cassette carrier.

2. Sealing and filling the Upper buffer chamber.

Wet the Upper buffer chamber (UBC) sealings with buffer solution or 0.1% SDS (immerse the sealings in solution or spray the sealings of the UBC using a plant sprayer) and carefully slide the UBC over the gel cassettes. Do not move the UBC repeatedly up and down as this will reduce the sealing effect. Fill the UBC with 1.2 liters of 2x buffer and use a funnel to adjust the buffer level in the lower buffer chamber to the same height as in the UBC, by adding 1x buffer.

3. Hydrostatic balance.

By adjusting the buffer level in the Lower buffer chamber to be the same as the level in the UBC, you achieve a status called ‘hydrostatic balance’, where there will be no overall change in the levels of either buffer chamber during electrophoresis. This will also help to improve runs and minimize any possible ‘leakage’ from the UBC. See Fig 9.


Fig 9. Adjusting the buffer level.

Applying equilibrated Immobiline DryStrip gels 

The DALT gel cassettes have a “longer” glass plate. The cassette should be laid on the bench with the longer glass plate down, and the protruding edge oriented toward the operator , refer to Fig 2.

Fig 2. Positioning an equilibrated Immobiline DryStrip gel on the DALT gel cassette.

1. Position the Immobiline DryStrip gel.
Dip the equilibrated Immobiline DryStrip gel in the SDS electrophoresis buffer (Table 4) to lubricate it.

Table 4. 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, with nine volumes of double-distilled water.
Store at room temperature, refer to section 6, Buffer recipes.

Place the strip with the acidic end to the left, gel surface up onto the protruding edge of the longer glass plate (Fig. 2)

2. Ensure Immobiline DryStrip gel has good contact
With a thin plastic ruler, gently push the Immobiline DryStrip gel down so that the entire lower edge of the Immobiline DryStrip gel is in contact with the top surface of the slab gel (Fig 3). Ensure that no air bubbles are trapped between the Immobiline DryStrip gel and the slab gel surface or between the gel backing and the glass plate.


Fig 3. Pushing the Immobiline DryStrip gel down to contact the gel slab.

3. Optional: Apply molecular weight marker proteins.
Best results are obtained when the molecular weight marker protein solution is mixed with an equal volume of a hot 1% agarose solution prior to application to the IEF sample application piece. The resultant 0.5% agarose will gel and prevent the marker proteins from diffusing laterally prior to the application of electric current. Other alternatives are to apply the markers to a paper IEF sample application piece in a volume of 15–20 μl. For less volume, cut the sample application piece proportionally. Place the IEF application piece on a glass plate and pipette the marker solution onto it, then pick up the application piece with forceps and apply to the top surface of the gel next to one end of the Immobiline DryStrip gel. The markers should contain 200–1000 ng of each component for Coomassie staining and approximately 10–50 ng of each component for silver staining.

4. Seal the Immobiline DryStrip gel in place.
Agarose sealing solution prevents the Immobiline DryStrip gel from moving or floating in the electrophoresis buffer. If using the Laemmli buffer system, see Table 5. Melt each aliquot as needed in a 100 °C heat block (each gel will require 1–1.5 ml). It takes approximately 10 min to fully melt the agarose.

Table 5. Agarose sealing solution.
(25 mM Tris base, 192 mM glycine, 0.1% SDS, 0.5% agarose, 0.002% bromophenol blue, 100 ml) 

	Final concentration	Amount
Laemmli SDS electrophoresis buffer (see Table 4.)		100 ml
Agarose (NA or M)	0.5%	0.5 g
1% Bromophenol blue stock solution	0.002% (w/v)	200 μl

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.
Store at room temperature.

Note: An ideal time to carry out this step is during Immobiline DryStrip gel equilibration.
Allow the agarose to cool until the tube can be held with your fingers (60 °C) and then slowly pipette the amount required to seal the Immobiline DryStrip gel in place, refer to Fig 4. Pipette slowly to avoid introducing bubbles. Apply only the minimum quantity of agarose sealing solution required to cover the Immobiline DryStrip gel. Allow a minimum of 1 min for the agarose to cool and solidify.


Fig 4. Sealing the Immobiline DryStrip gel in place using agarose sealing solution.

Note: Place the unit close to a sink for easy rinsing and draining. Connect the tubing leading to and from the heat exchanger to a thermostatic temperature controller such as MultiTemp III. Do not connect the heat exchanger to a water tap or any other coolant supply that does not have pressure regulation. Position a suitable power supply (e.g. EPS 601) conveniently close to the electrophoresis unit.

1. When preparing to run a gel in the Ettan DALTsix Electrophoresis Unit, insert the anode assembly in the tank and then fill the lower buffer chamber tank with 4.5 liters of 1x Laemmli SDS electrophoresis buffer (Fig 1), refer to section 6. Buffer recipes.


Fig 1. Filling the Ettan DALTsix electrophoresis unit with anode buffer.

2. Turn on the circulation pump by plug the leads attached to DALTsix base unit. Only perform this operation once the tank contains buffer.

3. Connect an external thermostatic circulator (e.g. MultiTemp III) and set the temperature to 25ºC for a day run and to 30ºC for an overnight run.

Table 6 lists the recommended conditions for running lab-cast gels using Ettan DALTsix system. Electrophoresis is performed at constant power in two steps. Stop electrophoresis when the dye front is approximately 1 mm from the bottom of the gel. Temperature control improves gel-to-gel reproducibility, especially if the ambient temperature of the laboratory fluctuates significantly. For best results, gels should be run at 25 °C. After electrophoresis, remove gels from their gel cassettes in preparation for staining or blotting.

Table 6. Recommended electrophoresis conditions for second-dimension vertical gels.

	Step	Current (mA)	Voltage (V)	Power (W)	Time (h)
Day-run 1 mm thick gel	1 2	10 40	80 500	1 13	1:00 4:30-6:00*
Day-run 1.5 mm thick gel	1 2	15 60	80 500	1 13	1:00 4:30-6:00*
Overnight-run 1 mm thick gel	1 2	10 12	80 150	1 2	1:00 15-17*,†
Overnight-run 1.5 mm thick gel	1 2	15 18	80 150	1 2	1:00 15-17*,†

* Continue the electrophoresis until the bromophenol blue reaches the end of the gel.
† To speed up the separation it is possible to change to day-run protocol when ever convenient.

Table 6 lists the recommended conditions for running DALT Gel 12.5 using Ettan DALTsix system. Electrophoresis is performed at constant power in two steps. Stop electrophoresis when the dye front is approximately 1 mm from the bottom of the gel. Temperature control improves gel-to-gel reproducibility, especially if the ambient temperature of the laboratory fluctuates significantly. For best results, gels should be run at 25 °C. After electrophoresis, remove gels from their gel cassettes in preparation for staining or blotting. Precast gels have a barcode, number, and gel percentage printed on them, which should be noted for orientation.

Table 6. Recommended electrophoresis conditions for second-dimension vertical gels.

	Step	Current mA/gel	Voltage (V)	Power (W/gel)	Time (h:min)
DALT Gel 12.5 (1 mm thick gel, set temperature to 25°C)	1 2	10 40	80 500	1 13	1:00 4:30-6:00*
Overnight runs (1 mm thick gel, set temperature to 30°C)**	1 2	10 12	80 150	1 2	1:00 15-17*

* Continue the electrophoresis until the bromophenol blue reaches the end of the gel.
** For the best possible resolution, faster separation time should be used. Use the faster protocol instead. Change to the day-run protocol when ever convenient.

Take out the gels from the refrigerator and keep gels at room temperature. Open the gel package and remove the gel. The DIGE gels come in glass cassettes and are ready to be used. Allow the gels to reach room temperature before use.

Loading of focused Immobiline DryStrips

1. Place the gels in the Ettan DALT cassette rack.

2. Briefly rinse the Immobiline DryStrips by submerging them in a measuring cylinder containing SDS electrophoresis running buffer for Ettan DALT.

3. Holding one end of the Immobiline DryStrip with forceps, carefully place the Immobiline DryStrip in-between the two glass plates of the gel. Using a thin plastic spacer, push against the plastic backing of the Immobiline DryStrip (not the gel itself) and slide the strip between the two glass plates until it comes into contact with the surface of the gel.

Note: The strip should just rest on the surface of the gel. Avoid trapping air bubbles between strip and the gel and avoid piercing the second-dimension gel with the strip.

Note: The acidic end of the Immobiline DryStrip should be on the right side of the gel when the shorter of the two plates is facing the user.

Note: The gel face of the strip must not touch the opposite glass plate.



4. Melt an aliquot of agarose overlay solution in a heating block or boiling water bath for each Immobiline DryStrips. Allow the agarose to cool slightly and slowly pipette the molten agarose solution, along the upper surface of the gel, up to the top of the glass plate. Take care not to introduce bubbles. Do not allow the agarose to solidify.

5. Once the agarose solution has completely set the gel should be run in the second dimension as soon as practically possible.

The second-dimension vertical gel must be ready for use prior to Immobiline DryStrip gel equilibration.

Equilibration is carried out in a two-step process using tubes and volume of equilibration solution as specified in the table 1 below.

Preparatory steps
1. Place the IPG strips in individual tubes, with the support film toward the tube wall.

2. Prepare an appropriate volume of SDS equilibration buffer solution (see Table 2 below) then measure into two equal volumes. Add DTT to one portion (100 mg per 10 ml) and iodoacetamide to the other (250 mg per 10 ml).

Equilibration
1. Add the appropriate volume of SDS equilibration buffer (+ DTT) to each strip. Cap or seal the tubes with flexible paraffin film and place them on their sides on a rocker for the equilibration process. Equilibrate for 15 min.

2. Pour off buffer from above step and add the appropriate volume of SDS equilibration buffer (+iodoacetamide) to each strip. Again cap or seal the tubes with flexible paraffin film and place them on their sides on a rocker for the equilibration process. Equilibrate for an additional 15 min.

Note: Be consistent with the timing of the equilibration steps.

Note: The subsequent steps of gel assembly, preparation of electrophoresis unit, insertion of the gel into the precast gel cassette, and melting of the sealing solution can be performed as the Immobiline DryStrip gels are equilibrating, as long as the timeframes above are adhered to.

Table 1. Suggested containers and volumes of equilibration solution.

Strip length (cm)	Container	Equilibration solution (ml)
7	Disposable, 15-ml conical tubes	2.5 - 5
11	25 × 200 mm screw-cap culture tubes	5 - 10
13	25 × 200 mm screw-cap culture tube	5 - 10
18	25 × 200 mm screw-cap culture tube, Equilibration tubes or Petri dish	10 - 15
24	Equilibration tubes or Petri dish	10 - 15

Table 2. 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**	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% (w/v)	00 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). 
** For preparing the Tris-HCl, pH 8.8 solution see Table 3.

Store in 20- or 50-ml aliquots at -20 °C.
Table 3. 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
HCI	-	adjust to pH 8.8
Double-distilled water	-	to 1 l

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

1. Carefully insert the electrophoresis gels (Fig 8) and fill any empty slots with DALT blank cassette inserts.


Fig 8. Inserting the cassettes into the cassette carrier.

2. Sealing and filling the Upper buffer chamber.
Wet the Upper buffer chamber (UBC) sealings with buffer solution or 0.1% SDS (immerse the sealings in solution or spray the sealings of the UBC using a plant sprayer) and carefully slide the UBC over the gel cassettes. Do not move the UBC repeatedly up and down as this will reduce the sealing effect. Fill the UBC with 1.2 liters of 2x buffer and use a funnel to adjust the buffer level in the lower buffer chamber to the same height as in the UBC, by adding 1x buffer.

3. Hydrostatic balance.
By adjusting the buffer level in the Lower buffer chamber to be the same as the level in the UBC, you achieve a status called ‘hydrostatic balance’, where there will be no overall change in the levels of either buffer chamber during electrophoresis. This will also help to improve runs and minimize any possible ‘leakage’ from the UBC. See Fig 9.


Fig 9. Adjusting the buffer level.

The DALT gel cassettes have a “longer” glass plate. The cassette should be laid on the bench with the longer glass plate down, and the protruding edge oriented toward the operator (Fig 5).


Fig 5. Positioning an equilibrated Immobiline DryStrip gel on the DALT Precast Gel Cassette.

1. Position the Immobiline DryStrip gel 
Dip the equilibrated Immobiline DryStrip gel in the SDS electrophoresis buffer (Table 4) to lubricate it. If using the DALT Gel 12.5, the diluted cathode buffer can be used to lubricate the strip.

Table 4. 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.

Place the strip with the acidic end to the left, gel surface up onto the protruding edge of the longer glass plate (Fig. 5)

2. Ensure Immobiline DryStrip gel has good contact
With a thin plastic ruler, gently push the Immobiline DryStrip gel down so that the entire lower edge of the Immobiline DryStrip gel is in contact with the top surface of the slab gel (Fig 6). Ensure that no air bubbles are trapped between the Immobiline DryStrip gel and the slab gel surface or between the gel backing and the glass plate.

Fig 6. Pushing the Immobiline DryStrip gel down to contact the gel slab.

3. Optional: Apply molecular weight marker proteins
Best results are obtained when the molecular weight marker protein solution is mixed with an equal volume of a hot 1% agarose solution prior to application to the IEF sample application piece. The resultant 0.5% agarose will gel and prevent the marker proteins from diffusing laterally prior to the application of electric current. Other alternatives are to apply the markers to a paper IEF sample application piece in a volume of 15–20 μl. For less volume, cut the sample application piece proportionally. Place the IEF application piece on a glass plate and pipette the marker solution onto it, then pick up the application piece with forceps and apply to the top surface of the gel next to one end of the Immobiline DryStrip gel. The markers should contain 200–1000 ng of each component for Coomassie staining and approximately 10–50 ng of each component for silver staining.

4. Seal the Immobiline DryStrip gel in place
The agarose sealing solution prevents the Immobiline DryStrip gel from moving or floating in the electrophoresis buffer. For precast DALT gels, the agarose blocks the narrow gap(s) between the gel edge(s) and the lateral spacer(s) to prevent leakage of the upper buffer. Prepare agarose sealing solution for DALT precast gels using the agarose sealing solution from the DALT Buffer Kit. If using the Laemmli buffer system, see Table 5. Melt each aliquot as needed in a 100 °C heat block (each gel will require 1–1.5 ml). It takes approximately 10 min to fully melt the agarose.

Table 5. Agarose sealing solution.
(25 mM Tris base, 192 mM glycine, 0.1% SDS, 0.5% agarose, 0.002% bromophenol blue, 100 ml)

	Final concentration	Amount
Laemmli SDS electrophoresis buffer (see Table 1.)		100 ml
Agarose (NA or M)	0.5%	0.5 g
1% Bromophenol blue stock solution	0.002% (w/v)	200 μl

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. Store at room temperature.

Note: An ideal time to carry out this step is during Immobiline DryStrip gel equilibration.
Allow the agarose to cool until the tube can be held with your fingers (60 °C) and then slowly pipette the amount required to seal the Immobiline DryStrip gel in place (Fig 7). Pipette slowly to avoid introducing bubbles. Apply only the minimum quantity of agarose sealing solution required to cover the Immobiline DryStrip gel. Allow a minimum of 1 min for the agarose to cool and solidify.


Fig 7. Sealing the Immobiline DryStrip gel in place on a DALT precast gel using agarose sealing solution.