Figure 10. Effect of trypsin on NLP formation.

NLP were prepared by adding 1 mM of CaCl₂, Na₂CO₃, and NaH₂PO₄ to DMEM containing the indicated amounts of serum, in the presence or absence of 0.5% trypsin, followed by incubation at 37°C for 1 day (A–D) or 3 days (E–H). Trypsin was shown not only to release the inhibition caused by FBS or HS, but also to produce a dose-dependent increase in the amount of precipitation that increased with the serum dosage. (I) Trypsin treatment on NLP as demonstrated by SDS-PAGE. NLP formed as in Fig. 9 from 3 mM each of the 3 precipitating reagents as well as 1% FBS in DMEM, either in the absence (lane 1) or presence (lane 2) of 0.5% trypsin. After overnight incubation of the 1 ml incubation mixture at 37°C, 100 µl was removed, pelleted by centrifugation, and washed twice in DMEM, and resuspended in 16 µl of 50 mM EDTA in double-distilled water for SDS-PAGE. Lane 3 contains 5 µg of trypsin, as control, which shows as a 25 kDa band. Note a prominent 72 kDa band and two fainter 54 kDa and 30 kDa bands associated with FBS NLP (lane 1), all of which disappears with trypsin treatment (lane 2).