Kta explorer fplc

Size exclusion chromatography (SEC) fractionation was carried out using an ÄKTA Explorer FPLC (GE Healthcare) placed inside a cold (4 °C) chamber. A Superdex 200 10/300 GL column (GE Healthcare) was used and samples were eluted with either 25 mM ammonium acetate (pH 8.5) or a Superdex 75 10/300GL with 20mM Tris 20mM NaCl (pH7,5) (for aggregated Aβ1-25), at a flow rate of 0.5 ml/min. Prior to injection, samples were centrifuged at 4°C 16,000 × g for 20 min and 0.5ml of sample supernatant was injected onto the column. Aggregated Aβ1-25 peptide was filtered using 0.22µm filter devices prior to injection to prevent from injecting any large, fibrillary aggregates. Peptide elution was detected by absorbance at 280 nm, 275nm and 215nm and 0.5 ml fraction volumes were collected. Eluted fractions were either used immediately or aliquoted (50ul) and stored at -80°C. Where indicated, samples volumes were concentrated approximately 10x in a speed vacuum. Aliquots (2 μl) of samples were used for MALDI-TOF/TOF MS (ABI 4800 model, Applied Biosystems) measurements. Matrix solution of α-cyano-4-hydroxycinnamic acid (7 mg/ml in ACN/0.1% TFA (1:1, v/v)) was used for sample deposition. The sample (1 μl) was mixed with 1 μl of matrix solution and then 1 μl of this mixture was deposited in duplicates on the target plate and allowed to air dry. Samples were analyzed in reflectron positive mode.