The reaction was started by the addition of lung-soluble supernate
(cytosol), and the increase in absorbance was measured spectrophotometrically at 340 nm using a Shimadzu UV-2401 PC spectrophotometer (Shimadzu, Columbia, MD).
2], (46 mmol/L), and the supernate
containing HDL is recovered by low-speed centrifugation.
5 mL of sample (UC 1 or UC 2 diluted 1:5 with saline/azide) or protein calibrator (A/G or UPL at 100 mg/L), and the mixture was centrifuged at 2500g for 5 min before removal of the supernate
and resolubilization of the pellet in 1 mL of 0.
In the chemical precipitation methods, the precipitation steps for prior isolation of HDL were done manually, whereas the cholesterol in the supernates
was measured on automated clinical chemistry analyzers.
The apo B-containing lipoproteins were sedimented by centrifugation (5 min), and the cholesterol content was measured in the supernate
with the CHOD-PAP method.
The abnormal precipitin arcs for albumin disappeared when antisera specific for human albumin or IgA ([alpha]-chain specific) were incubated for 37[degrees]C for 30 min with the purified IgA-type M-protein (at a ratio of 6:1 by volume) and supernates
were concentrated by ultrafiltration.
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After incubation, tissue suspensions were centrifuged at 1500 x gravity (g) for 10 min, supernates
were aspirated to waste, and pellets were resuspended in 1-35 mL of 2 M sodium hydroxide, depending on pellet volume.
The protein contents in supernates
were determined using Sigma Diagnostic reagents and procedures (Sigma Chemical Co.
For studies using the COBAS instruments, supernates
from the magnetic particle-treated samples were analyzed on a COBAS MIRA analyzer (Roche Diagnostics) with serum iron reagents from Thereto-DMA, Inc.
determined with the Dow enzymic method after precipitation of [Mn.
were combined and centrifuged at 40 OOOg for 30 min.