Measuring Interactions Between ECM and TGFB-Like Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 223

Fig. 2. Immunoprecipitation assay for examining release of matrix-bound LTBP1
and latent TGF β .

2.1. Preparation of 35 S-Labeled ECM from Calvarial Osteoblasts
1.

Fetal rat calvarial osteoblast (FRC) culture medium: To 500 mL minimal essential
medium, alpha modification ( -MEM) (Gibco Life Technologies, Gaithersburg,

MD) add 1.1 g NaHCO 3 (if medium is not provided with NaHCO 3 ), 10% heat-
inactivated fetal calf serum (FCS) (serum lots are individually checked from sev-
eral suppliers; heat-inactivate by heating serum to 56

α

C for 30 min), 2 m M
L -glutamine (LG) (commercially available as 100X stock; Gibco), and 100 U/mL
penicillin/streptomycin (P/S) (commercially available as 100X stock; Gibco).
After the cells become confluent, the serum concentration is reduced to 5% and
100

°

µ g/mL ascorbic acid and 5 m M β -glycerophosphate are added to the medium

(make each as a 100X stock in α -MEM, filter sterilize then make 5-mL aliquots