The controls were prepared as follows: 100 µl of enzyme preparation was added into 50 µl 1 M HCl, mixed well, and 100 µl of cold reaction buffer was added into the mixture.
An equal volume of RNase and DNase-treated, CsCl-purified phage preparation was added to an equal volume of disruption buffer (prepared by the addition of 7.2 μL 2-mercaptoethanol to 1 mL of GTC stock solution [22.5 mL 6 M guanidium thiocyanate solution (Sigma), 6.8 mL H2O, 1.76 mL sodium citrate (0.75 M), pH 7 and 2.64 mL 10% sarkosyl]).
Crude laccase preparation was added to the solution and the increase of absorbance at 420 nm was monitored with a temperature-controlled spectrophotometer U-30100, HiTokyo, Japan, Japan).
A 20 μL of enzyme preparation was added to 1 mL of 50 mM citrate phosphate buffer, pH 7.0, containing 0.5%% (w/v) CMC as the substrate.
The commercial enzyme preparation was added as a solution in phosphate buffer to substrate to afford a final concentration of 0.2 M.
Fifty microliters of single phage preparation was added and incubated at room temperature for 1 h.
Each preparation was added at a final concentration of 100 µg/ml to MDA-MB-231 breast cancer cell suspension before plating the cells, which were left to adhere to platelet-coated surfaces for 1 h.
For electroporation, 3 µl of a plasmid preparation was added to an aliquot of competent cells and submitted to 2,5 kV 600 Ohms and 25 µF using a Biorad electroporation apparatus.
100 µl of enzyme preparation was added into 100 µl of cold reaction buffer (50 mM HEPES-NaOH pH 7.0, 50 mM glucose-1-P, 2.5 mM AMP, 1.2 units of phosphorylase a from rabbit muscle), and was incubated at 30°C for 30 min. The reaction was terminated by adding 50 µl 1 M HCl, and mixed with 500 µl of dimethylsulfoxide.
0.15 ml of GBSS1 enzyme preparation was added into 0.15 ml of solution A (50 mM HEPES-NaOH pH 7.4, 1.6 mM ADP glucose, 0.7 mg amylopectin, 15 mM DTT), and incubated at 30°C for 1 hour (mixed every 10 min).
