Bio-Rad PROTEAN Plus Hinged Spacer Plates and Combs User Manual
Model 385 and 395
Gradient Former
Instruction Manual
Catalog Numbers
165-2000 and 165-2001
For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
Table of Contents
Page
Section 1 Introduction..........................................................................................1
1.1 Specifications ....................................................................................................2
Section 2 Pouring a Linear Gradient Gel ...........................................................2
2.1 Calculating the Chamber Volumes ....................................................................2
2.2 Pouring the Gel from the Top ............................................................................2
2.3 Pouring the Gel from the Bottom.......................................................................3
Section 3 Pouring an Exponential Gradient Gel ................................................4
3.1 Concave Exponential Gradient Gel....................................................................4
3.2 Convex Exponential Gradient Gel .....................................................................5
Section 4 Pouring Two Gradient Gels.................................................................5
Section 5 Pouring Multiple Gradient Gels in a Gel Casting Chamber .............6
Section 6 Gradient Former Care and Maintenance...........................................7
Section 7 Theory of Linear and Exponential Gradient Gels .............................7
7.1 Linear Gradients................................................................................................7
7.2 Exponential Gradients .......................................................................................7
Section 8 Protocols .............................................................................................10
8.1 Preparation of Stock Solutions for Laemmli SDS-PAGE Slab Gels ................10
8.2 Calculating the Volume of the Gel...................................................................11
8.3 Gel Preparation................................................................................................12
8.4 Linear Gradient Gels .......................................................................................13
8.5 Exponential Gradient Gels...............................................................................14
Section 9 Equipment and Accessories...............................................................15
9.1 Model 385 and Model 395 Gradient Former and Accessories .........................15
9.2 Additional Required Equipment ......................................................................15
9.3 PROTEAN®II xi and Mini-PROTEAN®II
Vertical Electrophoresis Cells and Accessories ...............................................15
9.4 Electrophoresis Chemicals...............................................................................17
Section 1
Introduction
The Model 385 and Model 395 Gradient Formers are primarily used to construct reproducible linear and exponential polyacrylamide gradient gels. Both gradient formers can also
be used to construct sucrose density gradients for ultracentrifugation or elution gradients for
column chromatography. To obtain optimal chromatography elution gradients, tubing with an
inside diameter • 1/16" must be used.
The Model 385 Gradient Former has a 30-100 ml capacity, making it ideal for the construction of one or two identical standard (16 x 20 x 0.15 cm) polyacrylamide gradient gels,
or up to 10 identical miniature (8 x 7 x 0.15 cm) gradient gels. The Model 395 Gradient
Former has a 100-700 ml capacity, making it ideal for the construction of up to 12 identical
standard (16 x 20 x 0.15 cm) gradient gels using a gel casting chamber.
The mixing and reservoir chambers of both gradient formers have identical dimensions.
The outlet from the mixing chamber leads through a peristaltic pump (not required for linear
gradients) to a single gel, to a Y for two gels, into a gel casting chamber for multiple gels. The
gradient may be cast from the top or bottom of the gel sandwich. When the gradient is formed
in a gel casting chamber, the gradient can be cast only from the bottom. Exponential pistons,
available as accessories for both gradient formers, are used to fix the volume in mixing chamber to produce concave or convex exponential gradients.
Fig. 1.1. Model 385 and 395 Gradient Formers, major parts.
1
Exponential piston
Reservoir chamber
Luer taper coupling
Mixing chamber
Peristaltic
pump
Valve stem
Stopcock
Tubing
Y-connector
1.1 Specifications
Model 385 Gradient Former
Overall size 10 cm x 15 cm x 15 cm (with piston in chamber)
Weight 405 g without piston; piston 65 g
Capacity 2 x 56 ml
Model 395 Gradient Former
Overall size 16.5 cm x 11.2 cm x 18 cm (without piston)
Weight 730 g without piston; piston 310 g
Chamber volume 2 x 400 ml
Practical capacity 2 x 350 ml
Construction of Model 385 and 395 Gradient Formers
Body machined from an acrylic block
Valve stem Delrin
®
Stopcock one-way plastic stopcock, Luer taper
Piston Delrin
Tubing Tygon
®
1/8" ID
Section 2
Pouring a Linear Gradient Gel
2.1 Calculating the Chamber Volumes
Each chamber's volume is one-half the total volume of the gel. The volume of the tubing
and the stir bar, however, can affect the measurement of the total volume. Section 2.2, step 10,
discusses tubing volume.
Adding the stir bar to the mixing chamber increases the volume in the mixing chamber.
If not compensated for, this increased volume would divide itself between the two chambers
when the valve stem was opened. However, when the stirring motor is turned on, a vortex is
created, which compensates for the increase in mixing chamber volume, since the bottom of
the vortex becomes the level at which the other chamber equalizes.
To calculate the volume of solution required for each chamber, multiply the spacer thickness in cm by the length of the gel in cm, by the width of the gel in cm, and divide by 2.
2.2 Pouring the Gel from the Top
The heavy solution is poured into the mixing chamber since it enters the sandwich first,
flowing to the bottom. The light solution, which flows into the gel last, is poured into the
reservoir chamber.
Note: We advise you to familiarize yourself thoroughly with the sequence of operations
starting with step 6. The best guarantee of reproducibility from gradient to gradient is
careful technique on the part of the operator.
1. Set up the gel sandwich in the usual manner.
2. Arrange the tubing in the peristaltic pump so there is as short a distance as possible
between the stopcock opening and the pump, and between the pump and the gel sandwich.
Cut the tubing as necessary.
3. Attach the needle to the end of the tubing and tape it to the top of the gel sandwich near
the center.
2
If a peristaltic pump is not used, the level of the gradient former stopcock must be above
the top of the gel sandwich by a distance sufficient to create a hydrostatic head large
enough to pour the entire volume of the gel within 10 minutes from the time the initiators
are added to the light solution. All the acrylamide should be in the gel sandwich before
polymerization begins, so that polymerization will be uniform throughout the gel.
Rather then using a needle on the end of the tubing, you may cut the end of the tubing at
an angle before taping it to the sandwich. Face the tapered opening toward the glass plate.
4. Place a 1" stir bar (Model 385) or 2" stir bar (Model 395) in the mixing chamber (with
valve stem closed).
5. Degas the heavy and light solutions.
6. Add the initiators to the light solution, swirl it 8 to 10 times, and pour it into the reservoir
chamber. (This is the start of the 10 minutes.) Leave the valve stem closed.
7. Add the initiators to the heavy solution, swirl it 8 to 10 times, and pour it into the mixing
chamber.
8. Start the stirring motor and adjust the speed so that you get good mixing and still the bot-
tom level of the vortex matches the acrylamide level in the reservoir chamber.
9. Quickly open the valve stem and stopcock, and turn on the peristaltic pump.
Note: The peristaltic pump must be able to pump the whole gradient within 10 minutes
from the time you add the initiators to the first solution. This is generally in the range of
5 to 10 ml/min for the Model 385 and 70 to 100 ml/min for the Model 395.
10. Run the pump until all the solution is pushed into the sandwich.
If a peristaltic pump is not used, the acrylamide will flow down the tubing to the gel sand-
wich by gravitational force when you open the stopcock.
Note: If tubing is left dangling below the top of the sandwich, you will have to lift it up
and drain it into the top of the gel sandwich to get the entire volume of acrylamide into
the sandwich. The tubing volume should not be a problem because the tubing will retain
a negligible amount of acrylamide following gradient formation.
11. Overlay the acrylamide with water or water saturated isobutanol. For a continuous buffer
system, insert a comb.
Note: Immediately after the gradient is cast, the system must be flushed with water to pre-
vent polymerization of residual acrylamide within the gradient former or pumping system.
12. Immediately remove the needle (or tubing) from the top of the sandwich and transfer to
a waste receptacle. Pour rinse water into both the mixing and reservoir chambers and turn
the peristaltic pump on to its maximum speed. The water should immediately flow from
the gradient former, through all connectors and tubing of the pumping system, and out the
needle (or tubing) to a waste receptacle.
2.3 Pouring the Gel from the Bottom
In this case, the light solution goes in the mixing chamber, and the heavy solution goes
in the reservoir. The light solution enters the gel from the bottom and travels to the top, and
the heavy solution follows, filling the bottom of the gel.
1. Set up the gel sandwich, gradient former, tubing, and peristaltic pump as described in
Section 2.2, steps 1, 2, 4, and 5. For a continuous gradient, insert the comb at an angle.
3
2. Insert the needle into the sandwich through the gasket on the bottom of the casting stand.
3. To start the linear gradient, use the procedure described in Section 2.2, steps 6-9, except
pour the light solution into the mixing chamber and the heavy solution into the reservoir.
4. After the stirring motor is turned on, the vortex level adjusted, the valve stem and stop-
cock opened, run the peristaltic pump until the last of the acrylamide enters the needle fit-
ting. Do not allow air bubbles to enter the gel, as this could cause the mixing of the
gradient. Remove the needle from the bottom of the casting stand.
5. Overlay the gel. For a continuous gel, the comb, which should be positioned at an angel
between the gel sandwich, is straightened and inserted fully to form the wells.
6. Rinse the residual acrylamide from the gradient former, tubing, and needle as described
in Section 2.2, step. 12.
Section 3
Pouring an Exponential Gradient Gel
In some cases, the number of proteins (polypeptides) in a certain area of the gel will justify pouring an exponential gradient. Whether or not a linear gradient gives adequate resolution is determined empirically for each sample. If there are many bands near the top of the gel,
then a concave gradient is indicated, whereas if there are many bands toward the bottom of
the gel, a convex gradient should be poured. (See Theory of Linear and Exponential Gradient
Gels, Section 7, especially Figures 7.3 and 7.5). The Model 385 gradient former piston (catalog number 165-2006) or the Model 395 gradient former piston (catalog number 165-2005)
limits the volume of the mixing chamber so that either a concave or a convex gradient can be
formed.
3.1 Concave Exponential Gradient Gels
Concave gradient gels are formed by delivering the acrylamide from the top, with the
small volume of heavy solution in the mixing chamber and the large volume of the light solution in the reservoir chamber. See Figure 7.2.
1. Calculate the chamber volumes.
Calculate the volume of the gel (spacer thickness in cm x gel width in cm x gel length in
cm). The volume of the heavy solution, for the mixing chamber, is one-fourth the total vol-
ume of the gel. The volume of the light solution, for the reservoir chamber, is equal to the
total volume of the gel. Because the volume of the mixing chamber is fixed, not all of
the gel solution will be delivered to the gel sandwich. One-fourth the volume will remain
in the mixing chamber at the end of the delivery and must be discarded.
2. Set up the equipment as in Section 2.2, steps 1-4.
3. Mix and degas the small volume of heavy acrylamide solution and the large volume of
light acrylamide solution, add the initiators to the light acrylamide solution, swirl 8 to 10
times, and pour into the reservoir chamber, keeping the valve stem closed.
4. Add the initiators to the heavy solution, swirl 8 to 10 times, and pour it into the mixing
chamber.
5. immediately fix the volume of the mixing chamber by inserting the piston into the cham-
ber to 1 cm above the level of the acrylamide and tightening the screw top handle to hold
the piston in place.
4
6. Quickly turn on the stirring motor (to a speed which gives good mixing without foaming),
open the valve stem and the stopcock, and start the peristaltic pump (see Note on the
speed of the peristaltic pump).
7. As the pump removes acrylamide from the mixing chamber, the reservoir solution will be
drawn into the mixing chamber and the gradient will be formed.
8. When the acrylamide solution reaches the desired level in the gel sandwich, stop the
pump, remove the tubing from the gel sandwich, and transfer it to a waste receptacle.
Overlay the gel with water or water-saturated isobutanol. For a continuous system, add the
comb to form the wells.
9. Remove the exponential piston from the mixing chamber and restart the pump to remove
any remaining acrylamide. When the chambers are empty, add rinse water to both the mix-
ing and reservoir chambers and flush out the system as described in Section 2.2, step 12.
3.2 Convex Exponential Gradient Gels
Convex gradient gels are formed by delivering the acrylamide from the bottom, with the
small volume of light solution in the mixing chamber and the large volume of heavy solution
in the reservoir chamber (see Figure 7.4).
1. Calculate the volumes of light and heavy solutions as in Section 3.1, except that the light
solution will be 1/4 the total gel volume and the heavy solution will be equal to the total
gel volume.
2. Set up the equipment as described in Section 2.3, with the needle entering the gel sand-
wich from the bottom through the casting stand gasket. For a continuous gradient, insert
the comb into the sandwich at an angle.
3. Mix and degas the small volume of light acrylamide solution and large volume of heavy
acrylamide solution.
4. Add the initiators to the light solution, swirl 8 to 10 times, and pour it into the mixing
chamber, leaving the valve stem closed.
5. Immediately fix the volume with the piston as described in Section 3.1, step 5.
6. Add the initiators to the heavy solution, swirl 8 to 10 times, and pour into the reservoir.
7. Cast the gradient as described in Section 3,1, steps 6-8.
8. Rinse out the system as described in Section 3.1, step 9.
Section 4
Pouring Two Gradient Gels
Double the volumes of heavy and light solution, and use the Y-connector provided. The
procedures are the same as above. It is essential that the tubing from the Y to the two gels be
the same length, and that the rate of flow be the same from each needle. We advise that this
be checked with water and two measuring cylinders ahead of time.
Note: It is difficult to attain reproducible gradients when pouring two gradient gels.
5
Loading...