β gal

Example 7

Cell lysates and ribosome samples were analyzed by ELISA according to the same steps described above for ribosome panning with the following modifications: (1) plates were coated with 100 μl of 10 μg/ml β-gal (Sigma) in PBS; and (2) 0.5% BSA was used instead of non-fat milk in the blocking solution. Following the washes after the samples were applied, instead of sample elution, 50 μl of anti-FLAG antibody (Stratagene) at a 1:5,000 dilution in blocking solution was added to each well and incubated for 1 h at 4° C. This was followed by four washes with cold washing solution and 1 h incubation at 4° C. with 50 μl of anti-mouse secondary antibody (Promega) at a 1:2,500 dilution in blocking solution. After four additional washes, bound scFvs were detected using SigmaFAST o-Phenylenediamine (OPD) tablets. The same procedure was followed for ELISA of protein samples, except that all steps were carried out at room temperature and in the absence of tRNA, heparin, and MgCl₂.

Example 3

Purified ubiquitin and ubiquitin activating enzyme (E1) were purchased from Boston Biochem, ubiquitin conjugating enzyme (E2, UbcH5a) was purchased from Millipore, Hsp70 was purchased from Enzo Life Sciences and β-gal was purchased from Sigma. Moreover, in vitro ubiquitination assays were performed in the presence of 20 mM MOPS pH 7.2, 100 mM KCl, 5 mM MgCl2 1 mM DTT, 4 mM ATP, 50 μM ubiquitin, 0.1 μM E1, 2 μM E2, 3 μM E3 (ubiquibody or control protein) and 3 μM target protein ((3-gal, gpD or Hsp70). Reactions were carried out at 37° C. and analyzed by SDS-PAGE and immunoblotting.