Dec 08, 2022
Canine/feline serum or plasma deproteinization for amino acid analysis on Biochrom
- Amanda Blake1,
- Jan Suchodolski1
- 1Texas A&M University - College Station
Protocol Citation: Amanda Blake, Jan Suchodolski 2022. Canine/feline serum or plasma deproteinization for amino acid analysis on Biochrom. protocols.io https://dx.doi.org/10.17504/protocols.io.877hzrn
Manuscript citation:
Blake, A.B.; Ishii, P.E.; Phillips, R.K.; Lidbury, J.A.; Steiner, J.M.; Suchodolski, J.S. Analytical Validation of an Assay for Concurrent Measurement of Amino Acids in Dog Serum and Comparison of Amino Acid Concentrations between Whole Blood, Plasma, and Serum from Dogs.Metabolites2022,12, 891. https://doi.org/10.3390/metabo12100891
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 11, 2019
Last Modified: August 08, 2023
Protocol Integer ID: 29663
Keywords: amino acids, deproteinization
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.
Materials
MATERIALS
750µL Nonsterile Micro-Centrifugal PVDF Membrane Filters, 0.2µm pore sizeThermo FisherCatalog #F2517-5
Amino acid standards physiological acidics and neutralsMerck MilliporeSigma (Sigma-Aldrich)Catalog #A6407
Amino acid standards physiological basicsMerck MilliporeSigma (Sigma-Aldrich)Catalog #A6282
L-NorleucineMerck MilliporeSigma (Sigma-Aldrich)Catalog #N8513-100mg
5-Sulfosalicylic acid dihydrate BioXtra >=99.0%Merck MilliporeSigma (Sigma-Aldrich)Catalog #S7422
Protocol materials
L-NorleucineMerck MilliporeSigma (Sigma-Aldrich)Catalog #N8513-100mg
5-Sulfosalicylic acid dihydrate BioXtra >=99.0%Merck MilliporeSigma (Sigma-Aldrich)Catalog #S7422
750µL Nonsterile Micro-Centrifugal PVDF Membrane Filters, 0.2µm pore sizeThermo FisherCatalog #F2517-5
Amino acid standards physiological acidics and neutralsMerck MilliporeSigma (Sigma-Aldrich)Catalog #A6407
Amino acid standards physiological basicsMerck MilliporeSigma (Sigma-Aldrich)Catalog #A6282
L-NorleucineMerck MilliporeSigma (Sigma-Aldrich)Catalog #N8513-100mg
5-Sulfosalicylic acid dihydrate BioXtra >=99.0%Merck MilliporeSigma (Sigma-Aldrich)Catalog #S7422
750µL Nonsterile Micro-Centrifugal PVDF Membrane Filters, 0.2µm pore sizeThermo FisherCatalog #F2517-5
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].
L-NorleucineMerck MilliporeSigma (Sigma-Aldrich)Catalog #N8513-100mg
5-Sulfosalicylic acid dihydrate BioXtra >=99.0%Merck MilliporeSigma (Sigma-Aldrich)Catalog #S7422
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.10000 x g
Expected result
You should expect a white pellet with some floating lipid debris and clear supernatant.
Transfer supernatant to centrifuge filter tubes, being careful not to disturb pellet or floating lipid layer.
750µL Nonsterile Micro-Centrifugal PVDF Membrane Filters, 0.2µm pore sizeThermo FisherCatalog #F2517-5
Centrifuge filter tubes at 10,000 rcf, 4°C for 5 minutes. 10000 x g Discard filter and store filtrate at —80°C until analysis.
Expected result
Very rarely, the filter will become clogged and you will need to transfer unfiltered liquid into a new filter on the same centrifuge tube and recentrifuge. You need at least 100 μl of filtrate to analyze but should aim for at least 250 μl filtrate to account for any necessary reruns.