Canine/feline serum or plasma deproteinization for amino acid analysis on Biochrom
Amanda Blake, Jan Suchodolski
Abstract
This protocol describes the deproteinization process that all canine and feline serum and plasma research samples undergo prior to amino acid analysis with a Biochrom 30+ Amino Acid Analyzer at the Gastrointestinal Lab, Texas A&M University. L-norleucine is used as an internal standard.
Steps
Aliquot 250 μl serum or plasma into 1.5 ml microcentrifuge tube.
Add 250 μl [5% Sulfosalicylic acid (w/v), 500 μM L-Norleucine] to 1.5 ml microcentrifuge tube. Note: if less than 250 μl serum or plasma is available, can use as little as 100 μl and add reagent in 1:1 (v/v) ratio. i.e., if using 150 μl serum, would add 150 μl [5% Sulfosalicylic acid (w/v), 500 μM L-Norleucine].
To make SSA solution, weigh 5 g sulfosalicylic acid into graduated cylinder, add 6.6 +/- 0.1 mg L-Norleucine, then fill to 100 mL with Biochrom lithium loading buffer. Stir briefly on magnetic stir plate until all solids are fully dissolved. Store at 4°C.
Vortex each on high speed for 5-10 seconds, and then store in 4°C for 10 minutes.
Centrifuge 1.5 ml tubes at 10,000 rcf, 4°C for 5 minutes.10000x g
Transfer supernatant to centrifuge filter tubes, being careful not to disturb pellet or floating lipid layer.
Centrifuge filter tubes at 10,000 rcf, 4°C for 5 minutes. 10000x g Discard filter and store filtrate at —80°C until analysis.
