The sections were then incubated in primary antibody diluted in blocking buffer overnight in cold room (4 °C), followed by three times rinse in PBS-T and 1 h incubation in secondary antibody in blocking buffer was followed by three times rinses in PBS-T.
For immunohistochemistry heat antigen retrieval in citrate buffer was followed by washing the sections in aqua dest.
Both the activities were followed in a buffer containing 5 µM of protein, MtbDGC, MtbPDE or MSDGC-1 and 25 mM Tris-HCl (pH 7.9), 250 mM NaCl, 10 mM MgCl2 in 50 µl volume.
Thereafter, washing in 1× PermWash buffer was followed by a 45-min incubation period at RT in 100 μL of a secondary solution containing 1 200 rabbit anti-mouse AlexaFlour488-conjugated Ab (Abcam) and 1 50 TO-PRO solution (Invitrogen, Carlsbad, CA, USA) in 1× PermWash buffer.
Application of cell lysate in binding buffer was followed by a washing step with 10 ml of binding buffer containing 60 mM imidazole and an elution step with 5 ml of elution buffer (50 mM Hepes/NaOH, pH 8.0, 300 mM NaCl and 500 mM imidazole).
Three washing steps with washing buffer (0.05% saponine/1% HSA in TBS) were followed by blocking with 250 μL blocking solution (5% donkey serum (Jackson immunoresearch) in washing buffer).
Standard operating procedures of the manufacturer were followed, applying all required buffers and solutions in the form provided by the manufacturerh.
Standard operating procedures of the manufacturer were followed, applying all required buffers and solutions in the form provided by the manufacturer (R&D Systems, Minneapolis, MN, USA).
The preparation and conditioning of DNA-modified electrodes, in which glassy carbon electrodes are covered by a thick film of double-stranded DNA and subsequently conditioned in acetate buffer, has been followed with Electrochemical Impedance Spectroscopy.
Purification and refolding of the peptides were accomplished by solublizing them with denaturants and dialyzing them in appropriate buffers, this being followed by chromatography of the refolded fractions on a metal-ion chelating column.
