Transfer of Proteins From Gel to Membrane

1. Wet nitrocellulose membrane in distilled water.

2. Presoak sponges, nitrocellulose membrane and Whatman 3MM paper in transfer buffer.

Fig. 2. (opposite page) Flow cytometric analysis of cell surface markers on purified follicular lymphoma cells. (A) Assessment of purity. Open histogram: immunostaining with a fluorescently conjugated isotype matched control antibody. Closed histogram: immunostaining with a fluorescently conjugated antibody against CD20, CD40 (B-cell markers), or CD3 (T-cell marker). Cell counts are shown on the j-axis, and fluorescence intensity is shown on the x-axis. Note that CD3-positive, CD40/CD20-negative T-cells are completely removed by the selection protocol leaving pure B-cells. (B) Assessment of clonality. Expression of the two immunoglobulin light chains kappa and lambda is normally approximately equal in a nonclonal population of B-cells, whereas clonal B-cell malignancies show clonal restriction and express a single light chain. Therefore, clonal restriction is a simple measure of the proportion of malignant cells in a B-cell preparation. Light chain expression was analyzed by double staining using FITC (fluorescein isothiocyanate) conjugated anti-kappa (x-axis) and PE (phycocoerythrein) conjugated antilambda (j-axis) antibodies (Dako, Ely, UK). Note that the negatively selected B-cells studied here are pure B-cells (all cells express light chains) and are clonal (all express kappa) with very few contaminating normal lambda-positive B-cells. See Color Plate 1, following page 270.

3. Place open cassette in a suitable container with enough transfer buffer so that it just covers the surface of the cassette.

4. Assemble the gel sandwich in the following order: sponge, Whatman 3MM paper, gel, nitrocellulose membrane, Whatman 3MM paper, sponge. Smooth out any air bubbles after addition of each layer and close the cassette.

5. Place cassette in the transfer apparatus ensuring that the nitrocellulose membrane is placed towards the anode.

6. Add the ice block for cooling, fill the tank with transfer buffer and run at 100 V for 1 h.

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