Step 3: Molecular-sieve chromatography. A sample of these concentrated active fractions (500 [[micro]liter]) was fractionated by molecular-sieve chromatography (gel filtration) (Superdex 75 HR10/30, prepack, Pharmacia).
The active fractions from molecular-sieve chromatography (fractions 6-8; [ILLUSTRATION FOR FIGURE 2A OMITTED]) were pooled and incubated for 15 min at temperatures from 4 [degrees] C to 100 [degrees] C, and were immediately returned to 23 [degrees] C.
Therefore, concentrated active fractions from molecular-sieve chromatography (6 and 7 in [ILLUSTRATION FOR FIGURE 2A OMITTED]) were electrophoresed on 15% SDS gel for a longer period (9 h), and were then transferred to a PVDF membrane.
Each fraction eluted in molecular-sieve chromatography [ILLUSTRATION FOR FIGURE 2A OMITTED] was concentrated, and the proteins were analyzed by SDS-PAGE.
Active fractions 6-8 from molecular-sieve chromatography [ILLUSTRATION FOR FIGURE 2A OMITTED] were pooled, and the thermostability of the OHSS solution was examined.
An antiserum raised from molecular-sieve chromatography (fractions 6 and 7) detected only two strong protein bands on SDS-PAGE, and they appeared in fractions 6-8 [ILLUSTRATION FOR FIGURE 5 OMITTED].
The activity eluted as a single peak on molecular-sieve chromatography, but still showed two protein bands at 32 kda and 30 kda on SDS-PAGE [ILLUSTRATION FOR FIGURE 2B OMITTED].
Thus, caseinolytic proteases are not present in the materials applied to the column for molecular-sieve chromatography [ILLUSTRATION FOR FIGURE 2A OMITTED].
As shown in Figure 2A, OHSS activity always appeared in fractions 6-8 on molecular-sieve chromatography. On SDS-PAGE, 32-kDa and 30-kDa protein bands appeared very clearly in these fractions [ILLUSTRATION FOR FIGURE 2B OMITTED].