Figure 2.
RNA gel retardation assay and SDS/PAGE analysis of the protein fractions obtained by RNA affinity chromatography. (A) After dialysis, aliquots (5 μl) of protein from each purification step were analyzed for RNA binding by a RNA gel retardation assay using [32P]-labeled transcript c. The RNA–protein complexes were then resolved in 4% native polyacrylamide gel. TE, crude testicular extract; FT, flowthrough fraction; W1-W4, wash fractions; E1-E3, 0.5, 1.0, and 2.0 M KCl step elution fractions, respectively. (B) Aliquots (30 μl) of the protein fractions analyzed in A were electrophoresed in SDS/12.5% PAGE and silver stained. Sizes of the prestained protein markers are indicated at right. Arrow in A indicates the location of the TB-RBP–transcript c complex in crude extracts. Arrowhead indicates the RNA–protein complex formed with transcript c and another testis RNA-binding protein (23).