At both time points, serum samples were analysed for 25D by competitive binding protein assay (Diasorin, Stillwater, Minnesota, USA), 1,25D by radioimmunoassay after immunoextraction (IDS, Tyne and Wear, UK), PTH by ELISA Scantibodies Laboratoryy, Santee, CA), and Ca and P by colorimetric assays (Roche diagnostics, Mannheim, Germany).
Serum samples were analyzed for 25-hydroxyvitamin D3 by competitive binding protein assay (DiaSorin, Stillwater, Minnesota, USA), 1,25-dihydroxyvitamin D3 by radioimmunoassay after immunoextraction (IDS, Tyne and Wear, UK), PTH by ELISA (Scantibodies Laboratory, Santee, CA, USA), and calcium (Ca) and phosphate (P) by colorimetric assays (Roche Diagnostics, Mannheim, Germany).
Protein concentration in the extracts was determined by a dye binding protein assay (Bio-Rad, NY, USA) with respect to a bovine albumin-globulin standard [27].
Hyaluronan concentration in joint fluid was measured by a sandwich binding protein assay using a Hyaluronate-Chugai test kit [ 33].
There is no guarantee that the DNA-binding specificity of the protein or DNA-binding domain assayed by SELEX is the same under the in vitro SELEX conditions as it is in in vivo.
SECIS binding of in vitro translated proteins was assayed by incubating 8 fmol with 20 fmol of wild-type or mutant (ΔAUGA) SECIS probes in 1 × PBS supplemented with 250 μg/mL yeast tRNA (Sigma), 10 mM DTT, and 5 μg soybean trypsin inhibitor (Sigma).
The fusion proteins were furthermore assayed by flow cytometry for binding to MUC1-expressing tumour cells.
Neither Saccharomyces cerevisiae CaM (ScCaM) nor a Ca2+ binding mutant of A. nidulans CaM (1234) interacts appreciably with A. nidulans CaM binding proteins by an overlay assay or activates two essential CaMKs, CMKA and CMKB.
Protein expression was assayed by western blot and Nf-κB DNA-binding activity by electrophoretic mobility shifts.
