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Ouchterlony Gel Diffusion

Background: 

Ouchterlony gel diffusion (a precipitation reaction) 

This is an example of a precipitation reaction between antibodies and soluble antigens, in this case, protein. The bivalent antibodies will cross-link the soluble antigen to form a lattice network and yield a visible precipitate. As with any antibody-antigen reaction, the antibody must specifically recognize the antigen. In this experiment, bovine serum albumin (BSA) will be used as the soluble antigen and anti-BSA antiserum as the antibody preparation. 

The Ouchterlony gel diffusion test is done in an agar gel prepared with cut out wells for the antigen and antibody. In today's experiment, the antibody is placed in the center well and varying concentrations of antigen are placed in the outer wells. The agar allows the antigen and antibody to diffuse freely out from the wells to establish a concentration gradient. The antibody is most concentrated close to the center well and becomes less concentrated further away from the well. This is also true for the antigen in the outer wells. At the points where the concentrations of both antigen and antibody are optimal, the lattice network will form into a visible band of white precipitate. These bands are immobilized in the gel. This procedure permits quantitative and qualitative characterizations of the antibody/antigen interaction. 

Procedure: 

1. The Ouchterlony plates are already poured and the wells have been punched out. In order to suck the plugs from the well, attach a Pasteur pipet to a vacuum flask. Carefully suck the plugs from the wells. Take caution to only remove agarose from the wells and not the surrounding area.

pasteur vacuum diagram

2. From the side bench, collect one tube containing antigen, (bovine serum albumin (BSA)) diluted 1:2. Collect a second tube containing antibody to BSA (a-BSA). Finally, collect a third tube containing sterile saline.

3. Prepare a serial dilution of antigen (BSA). Collect 6 Eppendorf (eppi or microcentrifuge) tubes and label them #1 through #6.

a. Dispense 50mL of saline (0.85% NaCl) into each of the 6 tubes labeled #1 - #6. Use aP200 pipetman. It is not necessary to change pipet tips between tubes as the solution is being dispensed into sterile, empty microcentrifuge tubes. 

b. Transfer 50 mL of antigen to tube #1. Draw the solution up and down gently 4-5 times in order to mix the solution and rinse the pipet. Do this gently to avoid making bubbles!!!!

c.Using a new pipet tip, transfer 50mL from tube #1 to tube #2. Mix as in step b.  

d.Using another new pipet tip, transfer 50 mL from tube #2 to tube #3. Mix. Proceed the same way until the last tube is mixed. Discard pipet tips appropriately. 

This series of two-fold dilutions will generate the following:

Tube: #1, #2, #3, #4, #5, #6 

Dilution: 1/4, 1/8, 1/161/32, 1/64, 1/128 

 

4. Label the bottom of an Ouchterlony plate; use the diagram below.

ouchterloney plate diagram

5. With aP20 pipetman, dispense 10 mL of the diluted antigen into the outer wells as labeled. If you start with the 1/128 dilution and continue to the 1/4 dilution you can use the same tip for all the antibody samples. 

6. With a clean pipet tip, fill the center well with 10mL of antibody (a-BSA).

7. Be careful not to disturb the fluid in the wells. Incubate, right side up, for 48 hours at room temperature in a humidified chamber (Tupperware with moistened paper towels in the bottom).

Results

Observe the precipitation on your ouchterloney plate. Yours will not be colored so be sure to look closely.

Ouchterloney plate results

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