More protein spots were detected when detergents were combined, i.e., 2% CHAPS, 1% Triton X-100 and 1%ASB-14ASB-14wed detectiof of up to 368 spots.
Soft tissues were removed from individual mice paws and homogenized in a solution containing 10 mM CHAPS, 1 mM phenylmethylsulphonyl fluoride (PMSF), 5 μg/mL, aprotinin, 1 μM pepstatin and 10 μM leupeptin.
Two hundred mg of starchy endosperm prepared from the opaque part of chalky grains or the corresponding part of perfect grains were ground in liquid nitrogen to fine powder and then suspended in 7 M urea, 2 M thiourea, 3%% (w/v) CHAPS, 1 % (v/v) Triton X-100, and 10 mM DTT.
Beads were washed twice with 0.5% CHAPS, 0.5% NP-40, and twice with 1% CHAPS, 1% NP-40.
The cell pellet was suspended in a lysis buffer containing 6 M urea, 2 M thiourea, 4% CHAPS, 1%DTT, 0.5% IPG buffer pH 4 7 (Amersham Biosciences).
Samples were diluted in isoelectric focusing buffer (7M urea, 2M thiourea, 4% CHAPS, 1% DTT) and absorbed into 17 cm ReadyStrip™ IPG strips (Biorad) following the manufacturer's directions.
Peptides were added in 10-fold molar excess compared to the 4A6 antibody, in a final volume of 380 µl of 1% CHAPS, 1% NP-40.
Standards of both Aβ40 and 42 were made in Antigen Capture Buffer (ACB; 20 mM NaH2PO4; 2 mM EDTA, 0.4 M NaCl; 0.5 g CHAPS; 1% BSA, pH 7.0), and loaded onto ELISA plates in duplicate.
Immobiline dry strips (pH range 3 10, 4 5.5, GE Healthcare) were rehydrated overnight in Rehydratation Solution containing 8 M urea, 2% CHAPS, 1% DTT and 1.5% IPG buffer pH 4 7.
After freezing-thawing cycles, sonication, and centrifugation, membrane-enriched protein fractions were resuspended in solubilization buffer (4% CHAPS, 1% Triton X-100), centrifuged at 10,000g, and protein concentration determined using the Micro BCA protein assay kit (Pierce).
A 100 mg portion of the liver was transferred into 1 ml of buffer (10 mM Hepes-KOH pH 7.4, 42 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.5% CHAPS, 1 µM PMSF, and 2 µg/ml leupeptin).
