2-D Immunoelectrophoresis

        Two-dimensional (2-D) immunoelectrophoresis, also known as crossed immunoelectrophoresis, is a particularly useful technique for the quantitation of one or more proteins in a mixture of proteins and the analysis of the    composition of protein mixtures. The method consists of two sequential electrophoretic steps: 

        1. The first dimension, during which the protein mixture to be analysed is separated by electrophoresis in an agarose gel. 

        2. The second dimension, during which the separated proteins are electrophoresed at right angles into a freshly applied layer of agarose containing a predetermined amount of antibody.


     A. Reagents and Equipment

         Reagents and reagent preparation

  • Electrophoresis buffer: 0.03 M barbitone buffer, pH 8.4. Take 5.15 g of sodium barbitone, 0.92 g barbitone, and make up to 1 L with distilled water (see Notes 1 and 2).
  • 1% Agarose in electrophoresis buffer. Fully dissolve the agarose by boiling for 2-3 min. This can be most conveniently done in a microwave oven. Care! When this boils, it can become very frothy and flow out the top of the flask. Ensure you have a small volume in a large flask, e.g., 50 mL in a 250 mL flask. When the agarose has dissolved, place the flask in a 55 oC water bath (see Note 3). Use agarose that has low electroendosmosis.
  • Bovine serum.
  • Reference serum: bovine serum containing one drop of concentrated Bromophenol blue solution.
  • Anti-bovine serum. 

        Equipment

  • Glass plates, 5 x 5 cm, 1.0-2.0 thick (see Note 4).
  • Flat-bed electrophoresis apparatus with cooling plate.
  • A microsyringe for loading samples in the wells.
  • A levelling plate.
  • Gel well-puncher: These are commercially available, although the author successfully uses a Pasteur pipette attached to a weak vacuum source.
  • Electrode wicks: These can be six thicknesses of filter paper, cut exactly to the width of the glass plate and soaked in electrophoresis buffer (see Note 5).
  • Protein stain: 0.1% Coomassie brilliant blue in 50% methanol and 10% acetic acid. Dissolve the stain in the methanol component first, and then add the appropriate volumes of acetic acid and water. 

     B. Method

  1. Dissolve the 1% agarose as described in Section A, and then place the flask in a 55 oC water bath. Stand a 10-mL pipette in this solution, and allow time for the agarose to cool to 55 oC At the same time, the pipette will be warming.
  2. Place a test tube in the 55 oC water bath, allow it to warm up for a few minutes and then add 5.5 mL of 1% agarose solution to the test tube. This transfer should be made as quickly as possible to avoid the agarose setting in the pipette. Briefly Whirlymix the contents and return to the water bath.
  3. Thoroughly clean a 5 x 5 cm glass plate with methylated spirit. When dry, put the glass plate on a levelling plate or level surface.
  4. Remove the test tube from the water bath and pour the contents of the tube onto the glass plate. Keep the neck of the tube close to the plate, and pour the contents of the tube onto the middle of the plate. Surface tension will keep the liquid on the surface of the plate. Alternatively, tape can be used to form an edging to the plate.
  5. Allow the gel to set for 5 min. While the gel is setting, pour 0.03 M barbitone buffer into each reservoir of the electrophoresis tank and completely wet the wicks. The wicks are prepared from six layers of Whatman No. 1 filter paper (but see Note 5). They should be cut to exactly the width of the gel to be run, and should be well wetted with barbitone buffer.
  6. Make two holes (1 mm diameter) in the gel 0.8 cm in from one edge of the plate and approx 1.5 cm in from the side of the plate. This is most easily done by placing the gel plate over a predrawn (dark ink) template, when well positions can easily be seen through the gel. The wells can be made using a Pasteur pipette or a piece of metal tubing attached to a weak vacuum source (e.g., a water pump).
  7. Place 0.5 pL of serum (bovine) in the test well and 0.5 pL of reference serum in the other well. "Top up" the wells with electrode buffer (~ 1 pL).
  8. Place the gel plate on the cooling plate of an electrophoresis tank, and place the electrode wicks over the edges of the gel. Take care not to overlap the sample wells, and ensure that these wells are nearest the cathode (see Notes 6-8).
  9. Immediately pass a current of 20 mA (~250 V) until the blue marker dye in the reference sample (which is bound to the albumin) reaches or just enters the anode wick. This will take approx 40 min. (This is about 10 V/cm across the plate.)
  10. Toward the end of this run, repeat step 2 using a new test tube, but this time use only 4.4 mL of agarose. (You will be covering a smaller surface of glass plate with this second dimension solution.)
  11. When the plate has run, return the plate to the levelling table, and cut out a gel slice which contains the test sample and not the reference sample. Transfer the gel slice to a second clean glass plate (see Note 9).
  12. To the tube in the water bath, now add 150 ~tL of rabbit anti-bovine serum, and briefly Whirlymix to ensure even dispersion of antibody. Return briefly to the water bath to allow any bubbles to settle out (see Notes 10 and 11).
  13. Pour the agarose and antibody mixture onto the second plate adjacent to the strip of gel, and allow the gel to set for 5 min.
  14. Place the plate in the electrophoresis apparatus as before and run at 3.0 mA/plate overnight (1 V/cm), making sure that the sample strip is at the cathode end (i.e., protein separated in the first dimension now moves toward the anode).
  15. At the end of the overnight run, precipitation peaks will be seen in the gel. These are not always easy to visualise and are best observed using oblique illumination of the gel over a dark background (e.g., using a dark background illuminator).
  16. A more clear result can be obtained by staining these precipitation peaks with a protein stain.

     C. Notes

  1. Barbitone buffer was the original buffer used for this method, but a suitable alternative is 0.1M Tris-borate, pH 7.4.
  2. The pH of the barbitone buffer is carefully chosen at 8.4, close to the isoelectric point of IgG. 
  3. Unused agarose solution can be stored at 4 oC and then reused at a later date. Each time the agarose is reused, check for signs of microbial contamination.
  4. The gel plates used should be as thin as possible to maximise the effect of the cooling plate. This is particularly important in the first-dimension run when considerable heat is evolved.
  5. Any highly absorbent, inert material can be used as the electrode wicks (e.g., filter paper, lint, cotton-wool). These wicks must be able to absorb a large amount of buffer and therefore have a low resistance. 
  6. The electrode wicks can "slip off" the gel during the course of an electrophoresis run. This can be prevented by placing thick glass blocks on top of the wicks, where they join the gel, or a heavy glass sheet across both wicks. 
  7. A uniform field strength over the entire gel is critical in rock immunoelectrophoresis. For this reason, the wick should be exactly the width of the gel.
  8. When setting up the electrophoresis wicks, it is important that they be kept well away from the electrodes.
  9. The working temperature for the agarose gel (55 oC) is just above the setting temperature of agarose. The agarose solutions must not drop below this temperature before pouring the gel. This temperature is chosen in order to minimise the chance of denaturing the antiserum, and the gel should be poured as soon as the antiserum has been added.
  10. When cutting the gel strip from the first dimension, make sure that sufficient gel remains adjacent to the sample track to allow for the placing on a wick on this gel in the second dimension. It is important that the wicks do not cover the sample when running the second dimension.
  11. The amount of antiserum to be used in the gel depends of course on the antibody titer and the amount of protein run in the first dimension, and is best determined by trial and error.
  12. The time quoted for electrophoresis is suitable for most samples. 
  13. If no results are obtained, check that the proteins being analysed run to the anode under the conditions used.