170 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS MATERIALS AND METHODS Antisera were prepared in rabbits against the alkali- and urea-extract- able proteins (hereafter called alkali and urea proteins, respectively), tonofibrin, and alkali-extractable proteins of the tissue residue after re- moval of tonofibrin (hereafter designated for convenience as tonofibrin- derived proteins) of normal human epidermis in the following way. The various proteins (4 ml) in barbital buffer (pH 8.6) and Freund's complete adjuvant (6 ml) were thoroughly emulsified by homogenization. Two milliliters of this mixture contained between 500 and 600 •g of protein and for each protein 2 ml were injected intradermally into some 30 sites on the back and sides of each of two rabbits. The rabbits were bled in 4 weeks and antisera were tested by precipitation of the antigens (im- munodiffusion). The next bleeding and the bleedout were completed in the next week and 2-week periods, respectively, provided the titers of the antisera remained high. If the antisera titers were low at the time of the first bleeding, the rabbits received further injections of the particular proteins after which the titers were determined after the second bleeding a week or two later. This procedure of reboosting was repeated several times in an attempt to obtain satisfactory titers. If the titers were not in- creased, the rabbits were bled out after one or two ear bleedings. The urea and alkali proteins which precipitated maximally at pH 5.5 were obtained from human epidermis as previously described (2, 4, 12). Briefly, dried, fat-free human epidermis was extracted with 6M urea in a Waring blender for 1.5 minutes (g of material X 50 -- ml 6M urea used). The extract was placed on a wrist action shaker at 0øC for 7 days. The extract was then stirred for 2 hours at room temperature (Magne stirrer). The mixture was then centrifuged at 1500 rpm for 10 minutes. The residue was washed once with 6M urea and again centri- luged. The supernatant and washing were combined and filtered through a sintered glass filter. The flitrate was dialyzed with stirring at 0øC against 20 volumes distilled H2 at pH 7.0-7.4 for 30 hours. The water was changed four times. The dialyzed urea extract was adjusted to pH 6.3 at which pH very insoluble proteins fiocculated. These proteins were separated by centrifugation at 18,000 rpm for 15 minutes at 0øC and discarded because of their insolubility. The supernatant was then ad- justed to pH 5.5. The proteins which precipitated at this pH were col- lected by centrifugation at 1500 rpm for 15 minutes and purified by re- peated solubilization in alkaline water, centrifugation, and precipitation

PROTE1NS FROM EPIDERMIS 171 at the isoelectric point. The supernatant fraction obtained from the pre- cipitation of the protein at pH 5.5 was then lowered to pH 4.5. At this pH a very small amount of other proteins flocculated, but the amount was too small for experimental use. In addition to the urea proteins which precipitated maximally at pH 5.5, the alkali proteins were obtained by extraction of the urea residue of epidermis with 0.05N NaOH by stirring (Magne stirrer or wrist action stirrer) at 0 ø for 7 days. The alkaline extract was diluted threefold with distilled water and stirred for 2 hours with a Magne stirrer at room tem- perature. The extract was passed through a sintered glass filter, and the pH of the flitrate was adjusted between 7 and 8. This mixture was then (entri[uged at 18,000 rpm for 20 minutes at 0øC to remove any insoluble materials. The pH of the supernatant was lowered to 6.3 with HC1. At this pH, very insoluble proteins fiocculated and these were separated by centrifugation at 18,000 rpm for 15 minutes at 0øC and discarded because of their inso]ubility. The supernatant was then adjusted to pH 5.5 and the proteins were allowed to fiocculate. The alkali proteins which pre- cipitated maximally at pH 5.5 were purified in the same fashion as the urea proteins of the same isoe]ectric point. Only small amounts of pro- teins which precipitated at pH 4.5 were obtained from the alkali extract of human epidermis. Both the purified urea and alkali proteins were solubilized in barbital buffer, pH 8.6. Tonofibrin and the alkali-soluble proteins were prepared in the following way. Dried and defatted human epidermal powder was ex- tracted with 75% LiBr for 48 hours at 25øC by the procedure of Roe (7). The resultant mixture was centrifuged and filtered through a coarse sintered glass filter. The flitrate was then dialyzed against distilled water (three changes including one overnight). A fibrous mass re- suited this was centrifuged and washed several times with distilled water. The tonofibrin was extracted from the washed fibrous tonofibrin by vigorous homogenization in barbital buffer, pH 8, followed by centrifuga- tion at 18,000 rpm for 15 minutes. The tissue residue remaining after the tonofibrin extraction was treated with 0.05N NaOH (25 ml per gram epidermis) in a closed recepticle by mixing in an electric shaker for 5 days at 25øC. The resulting extract was diluted threefold with distilled water and had a final pH of 7.7. This mixture was then stirred (Magne stirrer) for 2 hours, centrifuged at 1800 rpm for 20 minutes, and the supernatant passed through a course sintered glass filter. The pH of the tiltrate was adjusted to 8, and then centrifugation followed at 1%000 rpm