Spacer gel

The use of other coupling techniques with agarose is facilitated by the formation of agarose-spacer derivatives, thus increasing the versatility of the gel as a support in affinity chromatography. These derivatives are described in more detail in the Section on Spacer gels (p. 112). 

Fig. 16.1.Principle of epitope mapping using a two-dimensional electrophoresis procedure (proteins were dansylated only for visualization). The polypeptides are first separated by conventional SDS-electrophoresis (a) and a strip containing the separated polypeptides is embedded perpendicular to the direction of electrophoresis in a second gel (b). The protease, loaded on top of this second gel, is concentrated with its substrate (c) in the spacer gel through moving boundary electrophoresis. The peptides thus generated are separated in the resolving gel (d, e, f 1000, 100, and 10 ng total protein, respectively) and can be stained or transferred to nitrocellulose membranes (0,1 pm pores instead of the usual 0.45 pm should be used) and epitopes can be traced back to the various fragments of the same protein (from Tijssen and Kurstak, 1983 courtesy of Analytical Biochemistry).

For a protein separation, chloride and glycine are placed in the spacer gel and sample in the gel above it. How does the sample get between the chloride and the glycine and then what happens to it ... 

The spacer gel is large pore gel, i.e., it contains less crosslinking and has bigger holes. You will make enough of this for both the spacer gel and the sample gel. To store it, place it in one of the drawers by the apparatus to keep light off of it. Take a clean 10 milliliter stoppered, glass cylinder and add 1 mL of solution B. 

Use a syringe, and being really careful, add between 3-4 mm of the spacer gel to the tube so that it does not disturb the layer of the small pore gel. 

While the spacer gel is polymerizing, you should centrifuge your fish samples. Take the tubes of fish samples out of the ice bucket, put them in the centrifuge, turn the centrifuge on, and let them spin for 20 to 25 minutes. 

The sample gel is the same gel solution as the spacer gel, except that you mix your sample in with it. Take a 5... 

Now you can sit back and relax for a few minutes. In about 10 minutes you should see a little blue disc come down below the rubber stopper and go through the spacer gel. As soon as that blue disc gets to the small pour gel, raise the current to 5 miiliamperes per tube. It will take about 10 or 15 minutes for that disc to move down that far. Notice how beautifully sharp that disc will be. For some unknown reason, some students don t get the blue disc. If that should happen, then continue for 1.5 hours. Continue the separation until the blue disc gets to within 1 cm of the bottom of the tube. While this is developing, you will practice on the next technique. Get one of the tubes that was filled with gel but did not have a sample in it. 

The small pore separation gel on the bottom and the large pore spacer gel and a large pore sample gel on the top. 

The photopolymerization of the spacer gel is effected, the comb is carefully removed (in order not to destroy the surface of the wells), the plates are mounted into the electrophoretic apparatus, and samples are placed in the wells. After having filled the electrode vessels with buffer the electrophoretic separation can be started. Diffusion staining and destaining of the gel is done after disassembling the plates. 

Spacer gel is prepared from the above solutions by mixing them in a ratio... 

Remove the layer of distilled water over the resolving gel and pour the spacer gel. Insert at this time the comb chosen for the run. Allow the spacer gel to polymerize for at least 1 h but preferably overnight. 

Figure 2 Set-up for disk electrophoresis. (A) Tree-layer polyacrylamide gel ready for the run (B) isotachophoretic sample component stacking during the first few minutes of run (the process starts in the sample gel and has to be completed when leaving the spacer gel) (C) progress of separation in the running gel (Reproduced from Ornstein L (1964) Annals New York Academy of Sciences 121 321-349).

Immediately add the spacer gel solution up to the mark, 2.5 cm from the top, and overlay with distilled water. Leave the gel solutions to polymerize together for approximately 15 min. 

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