Figure 1

TAP Purification of RNA-binding protein complexes leads to RNA degradation when cells are lysed in a glass bead mill. A, 2 μl (corresponding to ~25 μg protein) of the input material for the IgG immunopurification (input) and the TCA-precipitated material from 200 μl of the TEV eluate from RJY358 (wt, untagged strain) and RJY929 (Nrp1p-TAP) were separated on a precast 4–12% gradient SDS polyacrylamide gel (Invitrogen) and stained with Coomassie. The molecular weight of the protein markers is indicated on the left. The band corresponding to the bait protein (Nrp1p-TAP) is labelled with an asterisk. B, 1 ml (corresponding to ~14 mg protein) of the input material for the IgG immunopurification (input) was phenol:chloroform extracted and 12 μg of the extracted RNA were separated on a 1.2% agarose-formaldehyde gel in the presence of ethidium bromide. As control for intact RNAs, 2 μg of total RNA (P/C lysis RNA) from the same strains prepared with a phenol method [18] were loaded in parallel as control. The position of the 18S and 25S ribosomal RNAs is indicated. C, crude lysate from strains RJY358 (wt, untagged) and RJY933 (Pbp2p-TAP) from two independent experiments were phenol extracted and 8 μg of the RNA were separated on a 1.2% agarose-formaldehyde gel and blotted onto a nylon membrane. 8 μg of total RNA (P/C lysis RNA) from the same strains prepared with a phenol extraction method [18] were loaded in parallel as control for intact RNAs. After methylene blue staining, the membrane was hybridized with a probe against PDA1 mRNA. The positions of the 18S and 25S ribosomal RNAs and the PDA1 mRNA hybridization signal are indicated.