The pH of aqueous phase was adjusted to 9 10 with 1 ml of 10 mol/l borate buffer, and this was followed by the addition of 500 μl of tert-butanol, approx. 3 g of anhydrous sodium sulfate, and 5 ml of MTBE.
The beads were washed and resuspended in SDS sample buffer; this was followed by an immunoblot analysis.
Extensive washing with streptavidin binding buffer (SBB) was followed by 30 min incubation with 2 mM biotin at 4°C.
Complex IV (cytochrome c oxidase) activity was determined by the change in absorbance at 550 nm (ε = 19.1 mM−1cm−1) due to the oxidation of reduced cytochrome c. 15 µM reduced cytochrome c and mitochondria were added to the assay buffer and the reaction was followed for 2 minutes.
Peptides were added to a 500 µl quartz cuvette containing 10 µg of LUVs in a HEPES 10 mM pH 7.4 buffer and the absorbance was followed during 20 min after peptide exposure.
Peptides were added to a 500 µl quartz cuvette containing 10 µg of LUV in a HEPES 10 mM pH 7.4 buffer and the absorbance was followed during 20 min after peptide addition.
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).
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.
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.
