PBCV lysin assays
PBCV lysin assays

A log phase NC64a culture (~500ml) was pelleted, washed 3x with BBM and suspended in 20 ml BBM, sodium azide was added to 0.02%. Assays employed 200ul of cell suspension and 2ul of purified PBCV (6/10/05) in a 0.5ml microfuge tube. After 30 min at 37C, 20ul of 10% SDS was added to lysin and a control (no PBCV). Samples were centrifuged 1 min at 5K. The control supernatant was clear (green pellet), the lysin supernatant was green (white pellet).

Neither PMSF, iodoacetamide nor EDTA inhibited lysin. 0.5mg/ml proteinase K had no lysin activity.

A further assay employed a control (no virus), standard lysin assay (w/virus), standard lysin w/1% Triton X-100 and standard lysin w/1% SDS. Following 30min @ 37C the first two were brought to 1% in SDS and all samples were centrifuged. Both "standard lysin" and 1% Triton samples showed lysis (green color). The latter showed much more red fluorescence (Triton enhances chlorophylll fluorescence). SDS, but not Triton inhibited lysin.

Based on these results the standard assay became - 100ul cells, 5ul 10% Triton X-100, 2ul virus, incubation at 37C followed by centrifugation for 30" at 5K.

Time series

Judging by green in pellets, longer digestions were more complete, but clearly 5' digests most cell walls

Vary detergent

Based on green color CTAB digestion was as good as Triton, but CTAB is ineffective in enhancing chlorphyll fluorescence.

Adding ammonium acetate (pH 7)

Lysin doesn't like ammonium acetate

Redox agents

Peroxide is no problem, but DTT is