Protein Standards Initiative

Protein Standard Initiative (PSI) http //psidev.sourceforge.net/... 

MUk protein standardizatiop (concentration of pasteurized skimmed milk). Milk protein standardization is designed to maintain the protein level in the milk constant all year round for automated cheese making. It basically involves concentration of pasteurized skimmed milk. It has been one of the major commercial successes in using inorganic membranes for food applications. In commercial production, microfiltration alpha-alumina and zirconia membranes with a pore diameter of 0.1 to 0.7 pm (mostly 0.2 pm) are used. Skimmed milk pasteurized at 70 C is typically concentrated to a volume concentration factor of 2 to 5 [Attia et al., 1988 van der Horst et al., 1994]. The volume concentration factor is the ratio of the initial feed volume to the retentate volume. Thus the higher the factor is, the more concentrated the product becomes. 

Figure 9.11 Release of bovine serum albumin from EVAc matrices. Controlled release of BSA from an EVAc matrix. Solid particles of BSA either (a) 45 to 75 /rm or (b) 150 to 250 /rm in diameter were dispersed within EVAc by solvent evaporation to achieve final protein loadings from 10, 20, 30, 40, or 50%. In each case five identical slabs, each 70 mg and 1 mm thick, were incubated in cacodylate-buffered water containing 0.02% gentamicin. Periodically, the buffered water was replaced, and the amount of protein released from the matrix was determined by measuring the concentration of protein in the solution that was removed. Each symbol represents the average cumulative fraction of protein released (cumulative mass of protein released/initial mass of protein within the matrix) for the five samples error bars indicate the standard deviation, which in some cases are smaller than the symbols. Data from [16].

Figure 15 Structure of an NBE-based (a) and COE-based (b) monolith. NBE/DMN-H6/2-PrOH/toluene/initiator=COE/TCOOMS/2-PrOH/toluene/ initiator=35 15 40 10 0.2. In both pictures, scale = 10 im. Separation of a protein standard on an NBE- (c) and a COE-derived monolith (d). Column dimensions (3 x 100 mm). Mobile phase A 95% water+ 5% acetonitrile+ 0.05% TEA mobile phase B 20% water+ 80% acetonitrile+ 0.05% TEA linear gradient, 22-80% B in 2.5 min, then 80% B up to 4.5 min flow rate = 3 ml min" f=25°C UV 200 nm. Peak order (1) lysozyme A, (2) ribonuclease, (3) insulin, (4) cytochrome c, and (5) myoglobin. Adapted with permission from Bandari, R. Prager-Duschke, A. Kiihnel, C. Macromoto/es 2006,...

Trial separations carried out with mixtures of protein standards are a practical way of getting started in HPIEC. Samples must be prepared, injected, collected and stored using techniques similar to those for a real-life sample. The added advantage with standards is that greater control exists over the composition and content of the sample. Furthermore, measurements of capacity, mass and activity recoveries are simpler and more accurate with known standard mixtures. In addition, the optimization of chromatographic parameters (Section 2.16) to maximize resolution, recovery and throughput, is best carried out initially using standard proteins of interest. 

Protein concentration can be determined using a method introduced by Bradford,4 which utilises Pierce reagent 23200 (Piece Chemical Company, Rockford, IL, USA) in combination with an acidic Coomassie Brilliant Blue G-250 solution to absorb at 595 nm when the reagent binds to the protein. A 20 mg/1 bovine serum albumin (Piece Chemical Company, Rockford, IL, USA) solution will be used to prepare a standard calibration curve for determination of protein concentration. The sample for analysis of SCP is initially homogenised or vibrated in a sonic system to break down the cell walls. 

The specifications and standardization include raw materials, preparation method of the standard solution, concentration of proteins, and the main band on SDS-PAGE. The outline of the procedure for preparation of the calibrators is shovm in Eig. 4.2. Table 4.5 shows the raw materials and the preparation method of the initial extract. To prepare the calibrators, the raw materials are extracted by the standard solution containing SDS and mercaptoethanol. The initial extract is prepared by centrifugation and filtration of the extract. The diluted extract is then prepared by 10-fold dilution of the initial extract with phosphate-buffered saline (PBS pH 7.4). The protein concentration of the diluted extract is assayed using the 2-D Quant kit (Amersham Bio Sciences). The standard solution is then... 

The protein content of the initial extract was determined using the 2-D Quant kit (Amersham Bio Sciences). The initial extract was diluted 20 times to make up the calibration standard solution. 

To measure the response of the biotinylated protein sample, add 3 ml of the (strept)avidin solution plus 75 pi of the HABA dye to a cuvette. Mix well and measure the absorbance of the solution at 500 nm. Next, add a small amount of sample to this solution and mix. Record the absorbance at 500 nm. If the change in absorbance due to sample addition was not sufficient to obtain a significant difference from the initial (strept)avidin-HABA solution, add another portion of sample and measure again. Determine the amount of biotin present in the protein sample by using the standard curve. The number of moles of biotin divided by the moles of protein present gives the number of biotin modifications on each protein molecule. 

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