Isolation of LacI-tagged SC35 from human SK-N-SH cells using dsDNA oligonucleotides. Total cellular lysates and isolated protein (PI) lysates were prepared from SK-N-SH cells transiently expressing LacI or LacI-tagged SC35. The protein isolation of LacI and LacI-tagged SC35 from PI-lysates was carried out using streptavidin beads pre-incubated with biotinylated dsDNA oligo specific for LacI (O-Sym). Western analysis of total cellular lysate (1), PI lysate (2), protein isolated by dsDNA oligos (oligo-PI) (3), and mock oligo-PI (4) from LacI–SC35 expressing cells, or PI lysate were generated from LacI expressing cells. Proteins were detected using either anti-Flag ( A ) or anti-SR protein (mAb 104) ( B ) antibodies. In addition, a Coomassie stained SDS–PAGE gel is shown as a qualitative comparison of dsDNA oligo PI versus immunopreciptiation (IP) using an anti-Flag antibody ( C ). In panel C: lane M, protein molecular weight marker; lane 1, 40 μg of total lysate; lane 2, oligo-PI; lane 3, Mock oligo-PI; and lane 4, anti-Flag IP. The asterisk marks the position of co-purifying immunoglobulin (i.e. heavy chain is shown) in the Flag-IP. The black arrows indicate the position of LacI–SC35 in panels B,C. Approximately 250–500 μg or 500–1000 μg of total protein lysate was used for western or Coomassie SDS–PAGE analysis, respectively.
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