Figure 3.

Recombinant GST–αCP₂ binds the 3′-UTR with a K_d of 2.1 nM. Recombinant GST–αCP2 was incubated at a constant amount, between 1 and 4 pmol, with increasing amounts of radiolabeled RNA probe added to the incubation mixture to determine the K_d of GST–αCP₂ binding. (A) In lane 2, 44 pmol αCP₂ was added to ensure that all the probe was active or able to be bound, while only 4.4 pmol αCP₂ was used in lanes 3–9. In lanes 1 and 2, 5 pmol radiolabeled RNA was used and in lane 3, 2.5 pmol was used. Then the amount of probe was increased to 5, 7.5, 15, 30, 46 and 60 pmol in lanes 4–9, respectively. The samples were run on a 6% native gel, dried and exposed on a PhosphoImager cassette and quantitated. (B) The equation shown was used in non-linear regression to determine the K_d of αCP₂ with the 3′-UTR of collagen α1(I) (19). Several gels were quantitated and analyzed separately and averaged to determine a K_d of 2.1 nM for αCP₂. (C) A Scatchard analysis of two representative gels was performed. Non-linear analysis was preferred to Scatchard analysis and this value was used for final determination of the K_d.