Protein calibration curves

FIGURE 4.2 Polyethylene oxide, dextran, and protein calibration curves for TSK-GEL SW Columns. Column TSK-GEL SW, two 7.S mm x 60 cm columns in series. Sample , proteins Q, polyethylene oxides O, dextrans. Elution dextrans and polyethylene oxides distilled water proteins 0.3 A1 NaCI in 0.1 M phosphate buffer, ph 7. Flow rate 1.0 ml/min. Detection UV at 220 nm and Rl. 

Figure 1. Protein calibration curves for the Spherogel TSK-SW 2000 and SW 3000 columns. Ten fiL containing 10--100 fig of each protein in 0,2M KPO buffer (pH 6.8) was chromatographed in the same buffer at 1.0 mL/min. Detection was at 254 nm X 0.16 AUFS pressure 500 psi. A chromatogram of 4 proteins on the SW 2000 column is also shown.

Figure 4.25 Protein calibration curves for Spherogel TSK-SN 2000 and TSK-SM 3000 colunn. Mobile phase phosphate buffer 0.2 M, pH 6.8, flow rate 1.0 sl/sin.

FIGURE 23.1 Example of a mustard seed protein calibration curve for a sandwich ELISA. (Courtesy of EUsa Systems.)... 

For the preparation of protein calibration curve, bovine serum albumin (BSA) was used. Different concentrations of BSA were prepared as 1 ml, and 2 ml of diluted Bradford reagent were added. The absorbance of these solutions was measured at 595 nm. 

The preliminary precipitation of proteins from milk is realized through the addition of solutions of acetic acid (1,7 mol/1) and sodium acetate (lmol/1) at t = 40-45°C before chromatographic isolation of OxTC. The precipitated proteins are separated by filtration. OxTC is detenuined in filtrate after its isolation on chromatographic column. Contents of OxTC was determined on calibration curve which is linear within concentration range 0,01-1,0 p.g/ml. 

FIGURE 2.4 Calibration curve of dextran on Sephacryi S-300 SF. Calibration curves were calculated from one chromatogram of a broad MWD reference sample using data for the molecular mass distribution as obtained by a calibrated gel filtration column ( , upper curve) and on-line MALLS ( ). The calibration curve was found useful for estimating the size of globular proteins. [Reproduced from Hagel et al. (1993), with permission.]... 

The ionic species of the mobile phase will also affect the separation. This is shown in Table 4.3 by the difference in resolution values for magnesium chloride buffer compared to sodium sulfate buffer. In addition, calibration curves for proteins in potassium phosphate buffers are shallower than those generated in sodium phosphate buffers. The slope of the curve in Sorenson buffer (containing both Na and ) is midway between the slopes generated with either cation alone (1). Table 4.4 illustrates the impact of different buffer conditions on mass recovery for six sample proteins. In this case, the mass recovery of proteins (1,4) is higher with sodium or potassium phosphate buffers (pH 6.9) than with Tris-HCl buffers (pH 7.8). 

TSK-GEL PW type columns are commonly used for the separation of synthetic water-soluble polymers because they exhibit a much larger separation range, better linearity of calibration curves, and much lower adsorption effects than TSK-GEL SW columns (10). While TSK-GEL SW columns are suitable for separating monodisperse biopolymers, such as proteins, TSK-GEL PW columns are recommended for separating polydisperse compounds, such as polysaccharides and synthetic polymers. 

FIGURE 4.43 Calibration curves for globular proteins on toyopearl resins. Column 22 mm X 30 cm. Sample Protein standards. Elution 0.06 A1 phosphate buffer, pH 7, in 0.06 A1 KCI. Legend elution volume V column volume. 

FIGURE 6.40 Calibration curves of Shodex PROTEiN KW-800 series. Column Shodex PROTEiN KW-802.S, KW-803, KW-804, 8.0 mm i.d. X 300 mm. Eiuent Pullulan, PEG PEO Purified water, Protein SO mM Sodium phosphate buffer + 0.3 M NaCI (pH 7.0). Flow rate 1.0 mL/min. Detector Pullulan. PEG PEO Shodex Rl Protein Shodex UV (220 nm). Column temp. Ambient. 

FIGURE 7.13 Preparative separation of various proteins on Fractogel EMD BioSEC (S). The length of the column was 1000 mm and the inner diameter 100 mm. The flow rate was 6.2 ml/min with 20 sodium phosphate buffer (pH 7.2) containing 0.3 M NaCI as the eluent. The injected standard proteins can be used to create a calibration curve. 

For proteins, the most useful columns are those with pores of 100-500 A, as seen in Fig. 10.2, because most proteins elute on the linear portions of the calibration curves. Figure 10.5 illustrates an analysis of a protein mixture on SynChropak GPC100. Small peptides can be analyzed on the 50-A SynChro-pak GPC Peptide column with appropriate mobile-phase modifications. Many peptides have poor solubility in mobile phases standardly used for protein analysis, as discussed later in this chapter. 

FIGURE 10.2 Calibration curves for proteins on SynChropak GPC columns. Mobile phase 0.1 M potassium phosphate, pH 7. (From MICRA Scientific, Inc., with permission.)... 

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. 

Proteins. Standard proteins for column calibration curves were obtained from Boehringer Mannheim. The methods outlined by Latham et al, (15) were followecLto prepare and label crude rat liver nuclear extract with -triiodothyronine. Normal con-... 

As a general rule, running blanks of the procedure highlights the level of contamination of amino acids. The evaluation of amino acid contamination (expressed in pgg 1 or mol 1) is possible by using calibration curves an estimation of three times this blank level may be considered as the lower limit to make a positive identification of protein... 

Comparative evaluation of anti-human thyroid stimulating hormone (hTSH) antibody, bound to the fifth-generation ammonia core (N5) or the fifth-generation ethylenediamine core (E5) dendrimer (1), did not show any differences in either the effective protein concentrations or the shape of the dose-dependent response curves (calibration curves) as determined from the recovery of standard controls. All the other experiments described here were thus carried out with the fifth-generation (i.e. dia. = 70 A) particles of ethylenediamine core (E5) de-ndrimers. The later particles were selected for their ability to be produced reproducibly on a large scale. 

Adrenal Tumours The assay-method is entirely based on the Schwartz-Mann Kit. According to this method, cortisol is first extracted from the plasma using CH2C12 (methylene chloride). In the actual radioimmunoassay the cortisol present in the extract competes with Cortisol-H3 i.e., the radioactive tracer) for the common binding sites on transcortin, which is incidently not an antibody but a cortisol-binding protein. Now, the free cortisol is quantitatively removed by adsorption on dextran-coated charcoal from the one bound to the transcortin. Finally, the bound radioactivity (due to Cortisol-H3) is measured which is then employed to calculate exactly the amount of cortisol present in the sample by the help of a Standard Curve (or Calibration Curve). 

The method is more sensitive than the biuret method and has an analytical range from 10 ju,g to 1.0 mg of protein. Using the method outlined below this is equivalent to sample concentrations of between 20 mg l-1 and 2.0 g l-1. The relationship between absorbance and protein concentration deviates from a straight line and a calibration curve is necessary. The method is also subject to interference from simple ions, such as potassium and magnesium, as well as by various organic compounds, such as Tris buffer and EDTA (ethylenediamine-tetraacetic acid). Phenolic compounds present in the sample will also react and this may be of particular significance in the analysis of plant extracts. 

The method is capable of detecting as little as 5 pig protein and a calibration curve is necessary because of the variations between different proteins and the non-linearity of the absorbance-concentration relationship. 

Figure 4.20 Calibration curves for size-exclusion liquid chromatography. Column, TSK GEL G3000SW, 120 cm x 7.5 mm i.d. eluent, 0.2 m sodium phosphate buffer pH 6.8 flow rate, 1.0 ml min-1. Standards 1, protein-, 2, dextran, and 3, polyethylene glycol.

A standard Lowry-based protein assay has been adjusted to the special conditions encountered with skin [126], Basically, proteins reduce an alkaline solution of Cu(II)-tartrate to Cu(I) in a concentration-dependent manner. Then, the formation of a blue complex between Folin-Ciocalteau reagent (a solution of complex polymeric ions formed from phosphomolybdic and phosphotungstic heteropoly acids) and Cu(I) can be measured spectrophotometrically at 750 nm. A calibration curve can be obtained by dissolving known amounts of stratum corneum in 1 M sodium hydroxide. A piece of tape that has not been in contact with skin is subjected to an identical procedure and serves as negative control. The method was recently adapted to a 96-well plate format, notably reducing analysis times [129],... 

Figure 7.3 Calibration curves of proteins using a 0.2 M sodium phosphate (pH 7.5) mobile phase. (Printed with permission of Agilent Technologies, Inc.)...

Protein content Bradford s test was performed to determine the protein content (mg g ) of the lyophilisates. A sample of the lyophilisate (2.5 mg) was dissolved in glycylglycine (Gly-Gly) buffer (1.5 mL). An ahquot of this solution (20 gL) was diluted with glycylglycine (Gly-Gly) buffer (30 gL) and Bradford s solution (950 gL) was added. After 5 min, the absorbance was measured at 595 nm. The concentration of FSA is calculated from the interpolation of the calibration curve of bovine serum albumin (BSA) 437 mg total protein/gram of lyophihsate. 

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