AOAC SPIFAN WPC ERP-Final Action Review (Nov. 2017)

2016.15 (Nov. 2017) - WPC-31 MLT-Final Action Review

FOR ERP USE ONLY DO NOT DISTRIBUTE

factor for whey proteins vs caseins in the calculation of whey protein content.

B. Apparatus a) ProteomeLab PA 800 plus, (Beckman Coulter, Inc., Fullerton, CA or equivalent), equipped with UV detector set at 220nm. Peak area can be integrated using any suitable software (Waters Empower, Beckman 32Karat, or equivalent). b) Bare, fused-silica capillaries of 50 μm ID × 30 cm with effective length of 20 cm (338451 from Beckman Coulter, Inc., Fullerton, CA or equivalent). c) Analytical balance: 4 decimal places. d) Temperature controlled water bath: 100±5 o C. e) Micro centrifuge for 1.5-mL vials. f) Vortex. C. Reagents a) SDS-MW gel buffer (A30341, Beckman Coulter, Inc. Fullerton, CA, recipe is readily supplied by the Vendor). b) SDS-MW analysis kit (390953 Beckman Coulter, Inc., Fullerton, CA) including: bare, fused- silica capillaries of 50 μm ID × 30 cm; SDS-MW gel buffer; SDS-MW sample buffer (100 mM Tris-HCl, pH 9.0, with 1% SDS); 10 kDa protein internal standard; acidic wash solution (high-purity 0.1 M HCl); basic wash solution (high-purity 0.1 M NaOH); and SDS-MW Size standard (10-225 kDa, 16 mg/mL). Contents of the kit can be replaced by equivalent sources. c) 10 kDa protein internal standard, (A26487, Beckman Coulter, Inc. Fullerton, CA). d) Water (LC grade). e) β-mercaptoethanol (M7154 or M6250 Sigma). D. Preparation of the system buffer trays (Beckman) as well as standard and sample solutions a) Load the reagents into the system inlet (left) and outlet (right) using 6x6 buffer trays arranged according to the configuration illustrated in Figure 2016.15A (buffer tray configuration). If using an equivalent system, follow the instructions for that system. b) Weigh either 135±5 mg skim milk powder (SMP, with protein content around 37%) or 500±20 mg of infant formula powder (with protein content around 11%) into a 10-mL centrifuge tube. c) Disperse the sample to 5 mL final with DI water. Vortex each tube until the solution appears homogeneous. Each final solution contains about 10 to 15 mg/mL of protein. d) Prepare the sample running pre-solution: mix the 1% SDS sample solution with 10 kDa internal standard peptide using an 84:1 ratio. The total volume of the pre-solution is based on the total number of samples to be analyzed in the sample set (90 μL per sample). e) Pipette 10 μL of each sample solution into separate 2.0-mL microcentrifuge vials. g) Adjustable pipettes: 20, 100, 1000 μL (Eppendorf or equivalent). h) 2.0-mL safe-lock centrifuge tubes (e.g. Eppendorf 022363344). i) 10-mL round-bottom centrifuge tube (e.g. Eppendorf 022822179).