Cation exchange - PBCV1 WAL lysin - 4
Prep 3, 0.5ml (1.0M LiCl wash) was diluted 3x. 15 microliters of 2.5mg/ml polyethyleneimine was added. The flocculant precipitate was removed by centrifugation and the supernatant was applied to Acrosep S column equilibrated w/0.4M NH4Ac. The column was eluted with 0.4, 0.6, 0.8, and 1.0 M NH4Ac.
Activity elutes at 0.6 - 1.0 ammonium acetate, i.e strongly bound
Fractions were precipitated with TCA, dissolved in 25 microliters of borate cracking buffer containing 2mM iodoacetamide, heated 4 min at 100C. Aliquots were applied to 4-20% polyacrylamide gels (SDS). Gels were stained with silver
Note - "S" is starting material
Again fractions with lysin activity seem to have multiple subunits
Note the smear of DNA above the protein bands has been eliminated (PEI ppt.)
