A flow rate of 72 ** f was used and 12 ml fractions were collected. Approximately 165 fractions were obtained from each column. These were read at 280 m | m in a Beckman model DU spectrophotometer and tested for antibody activity as described above.

For protein identification, fractions from the column were concentrated by pervaporation against a stream of air at 5 ` C or by negative pressure dialysis in an apparatus which permitted simultaneous concentration of the protein and dialysis against isotonic saline. During the latter procedure the temperature was maintained at 2 ` C by surrounding the apparatus with ice. Because negative pressure dialysis gave better recovery of proteins, permitted detection of proteins concentrated from very dilute solutions and was a gentler procedure, it was used in all but the earliest experiments.

Paper electrophoresis was carried out on the concentrated samples in a Spinco model R cell using barbital buffer, pH 8.6, ionic strength 0.075, at room temperature on Whatman 3 MM filter paper. Five milliamperes/cell were applied for 18 hr, after which the strips were stained with bromophenol blue and densitometry was carried out using a Spinco Analytrol.

When paper electrophoresis was to be used for preparation, eight strips of a whole serum sample or a chromatographic fraction concentrated by negative pressure dialysis were run/chamber under the conditions described above. At the end of the run, the strips in the third and sixth positions in each chamber were dried, stained for 1 hr, washed and dried, while the other strips were maintained in a horizontal position at 1 ` C. The unstained strips were then marked, using the stained ones as a guide, and cut transversely so as to separate the various protein bands. The strip sections containing a given protein were pooled, eluted with 0.5 ml of isotonic saline, and the eluates were tested for antibody activity.