The tissues were homogenized in 10% chilled Tris hydrochloride buffer (10 mM, pH 7.4) by tissue homogenizer (Remi Motors, India) and centrifuged at 12000 rpm for 15 min 0°C using Eppendorf's 5810-R high speed cooling centrifuge.
Protein was extracted in 4 M guanidine hydrochloride buffer containing 50 mM sodium acetate, pH 5.8, 1 mM phenylmethylsulfonyl fluoride, and 1 μg/ml aprotinin at 4°C and dialyzed.
Tissue was rinsed and reacted in diaminobenzidine (DAB, 0.5 mg/ml, Sigma) in a tris-hydrochloride buffer (Trizma, Sigma; pH 7.2) with hydrogen peroxide (0.35 μl 30% hydrogen peroxide/ml buffer).
Briefly, cell lysis was obtained by a guanidine hydrochloride based buffer.
The assay contained 1 mM NAD+, 50 mM Na2HPO4, 0.2 mM EDTA, and 0.5 mM glyceraldehyde 3-phosphate in 50 mM triethanolamine hydrochloride (TEA) buffer pH 8.5.
The column fractions were analyzed by SDS PAGE, and the desired fractions were combined before dialysis at 4 °C against 6 L OF triethanolamine hydrochloride (TEA) buffer (20 mM TEA (7.5) and 100 mM NaCl).
Bacterial pellets were suspended in 6 M guanidinium hydrochloride containing buffer, and the recombinant proteins were purified by nickel column chromatography according to manufacturer's instructions (Qiagen, Valencia, CA, USA).
As mentioned above, the MolYsis protocol uses a guanidine hydrochloride chaotropic buffer for lysis of human/animal cells, and bacteria with a thin or labile cell wall (e.g. members of the genus Treponema) or those exposed to cell wall-active antibiotics and/or human immunosystems or bacteria even devoid of a cell wall (e.g. Mycoplasma, Chlamydia) are also lyzed.
Ion-exchange HPLC was performed on a Jasco LC-900 HPLC system equipped with a Jasco UV-970 detector (detection at λ=254 nm) and a Dionex BioLC DNAPac PA-100 column (250×9 mm) with water/0.25 m tris(hydroxymethyl)aminomethane hydrochloride (Tris⋅HCl) buffer (pH 8)/1 m sodium chloride solution as the eluents.
After incubation, samples were centrifuged and the supernatant was purified using a 5 M guanidine-hydrochloride binding buffer with High Pure Viral Nucleic Acid Large Volume kits (Roche, Switzerland).
The mucus samples removed after thickness measurements were solubilized in a guanidinium hydrochloride-based buffer and processed by the FASP method as described before.
