Journal:

Article Title: The function of Xenopus Bloom's syndrome protein homolog (xBLM) in DNA replication

doi:

Figure Lengend Snippet: Characterization of xBLM protein. (A) Coomassie blue staining of the purified His-tagged recombinant xBLM protein. (B) Helicase activity of the recombinant xBLM. The substrate is a 5′-32P-labeled 48mer oligonucleotide hybridized to m13mp18 single-stranded DNA. The purified xBLM protein was incubated with the substrate in the presence of either buffer alone or nucleotides (ATP or dATP). The right lane contains the heat-denatured substrate. The dissociated oligonucleotide was separated from m13 on 12% polyacrylamide gels. (C) Western blot of interphase egg extracts (2 μL) probed with the affinity-purified rabbit anti-xBLM antibody and detected by ECL (Amersham). The 160-kD band is xBLM. The small band is not always detected, suggesting that it is a degradation product. (D) Immunodepletion of xBLM from egg extracts. Supernatants (2 μL) from the extracts that had been depleted with either anti-xBLM or control antibodies were analyzed by Western blot with the affinity-purified rabbit anti-xBLM antibody. Molecular markers are labeled as kilodaltons on the right side of panels A, C, and D.

Article Snippet: Immunodepletion was performed by incubating extracts (diluted twofold with egg lysis buffer) with Affi-gel Protein A beads (Bio-Rad) that had been precoated with either the affinity-purified rabbit anti-xBLM antibody or rabbit IgG.

Techniques: Staining, Purification, Recombinant, Activity Assay, Labeling, Incubation, Western Blot, Affinity Purification