For extraction, 2.5 g of oil was rotated end over end with 2.5 mL of heated (60 °C) RIDASCREEN® Allergen extraction buffer for 20 min at 60 °C.
In this test Chlamydia antigen is extracted from the specimen by heating the swabs at 80°C with the Extraction Buffer.
Extraction buffer was added in each extract before heating.
Each extract was diluted 1 4 in extraction buffer and an aliquot of 25 μL was injected into a C18 protein and peptide column heated to 55°C in a Waters 2695 Separation Module, and absorbance at 200 nm was measured with a Waters 2487 Dual Absorbance Detector.
Protein extracts were obtained using Cell Extraction Buffer buffer (BioSource International, CA, USA) plus protease inhibitor cocktail (Sigma).
Extracts were heated for five minutes at 60°C and 20 μl of assay buffer (containing Tris (hydroxymethyl) aminomethane 3.0% and BSA 0.10%) was added followed by the extraction buffer (to neutralise the extracts).
Cells were washed three times with PBS, lysed with 80 μL of extraction buffer, freeze/thawed in liquid nitrogen and heated at 85°C for 40 min.
Bacterial pellets of about 60 mg (wet weight) were heat inactivated for 1 h at 95°C in 500 µL extraction buffer (50 mmol/L Tris-HCl, 25 mmol/L EDTA, 5% monosodium glutamate) and sequentially treated with lysozyme (2 h, 37°C, 17 M lysozyme) and proteinase K (overnight, 45°C, 0.3 M proteinase K in proteinase K buffer: 1 mmol/L Tris-HCl, 5 mmol/L EDTA, 0.05% sodium dodecyl sulfate [SDS], pH 7.8).
Bacterial pellets of about 60 mg (wet weight) were heat inactivated for 1 hour at 95°C in 500 μl extraction buffer (50 mM Tris-HCl, 25 mM EDTA, 5% monosodium glutamate), and sequentially treated with lysozyme (2 h, 37°C, 17 M lysozyme) and proteinase K (overnight, 45°C, 0,3 M proteinase K in proteinase K buffer: 1 mM Tris-HCl, 5 mM EDTA, 0,05% SDS, pH7.8).
Extract peptides with 50 100 µl of extraction Buffer I (50% ACN, 0.5% FA) and then with 50 100 µl of extraction Buffer II (75% ACN, 1% FA).
