Purification and Assay of GFP-BLM. A. Fluorescent microscope image of GFP-BLM in the DAPI-stained nucleus of an Sf9 cell 72 hours after infection with a recombinant baculovirus. GFP-BLM forms large green aggregates. B. Silver-stained SDS 4-12% polyacrylamide gel of the purification of GFP-BLM using Macroprep Q ion exchange chromatography followed by metal chelate resin chromatography. Lane Q shows the ion exchange column pool of the green GFP-BLM fractions and lanes W1-W3 are proteins eluted from the metal resin with increasing imidazole washes (wash1 = 10 mM, wash2 = 50 mM, wash3 = 100 mM). The elution lanes show purified GFP-BLM eluted with a final step of 200 mM imidazole. The molecular weights (MW) of the standard markers (BioRad Precision) are indicated. C. Autoradiogram of a thin-layer plate ATPase assay of GFP-BLM. The amount of protein in the assay is shown. The addition of single-stranded Mpl8 DNA is indicated. Calf intestinal phosphatase (CIP) is used as a positive control for [γ-32P]ATP hydrolysis. D. Unwinding of a 32P-labeled 54 base oligonucleotide annealed to Mpl8 DNA by GFP-BLM. The position of the substrate (S) and the product (P) on the autoradiogram of a 10% polyacrylamide gel are indicated. The gel shows reactions containing 25 ng of GFP-BLM incubated for increasing time at 27°C and unwinding by increasing amounts of GFP-BLM incubated at 27°C for 30 minutes.
Yankiwski et al. BMC Cell Biology 2001 2:11 doi:10.1186/1471-2121-2-11