Protein standards

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. 

Fig. 3. Cation-exchange chromatography of protein standards. Column poly(aspartic acid) Vydac (10 pm), 20 x 0.46 cm. Sample 25 pi containing 12.5 pg of ovalbumin and 25 pg each of the other proteins in the weak buffer. Flow rate 1 ml/min. Weak buffer 0.05 mol/1 potassium phosphate, pH 6.0. Strong buffer same +0.6 mol/1 sodium chloride Elution 80-min linear gradient, 0-100% strong buffer. Peaks a = ovalbumin, b = bacitracin, c = myoglobin, d = chymotrypsinogen A, e = cytochrom C (reduced), / = ribonuclease A, g = cytochrome C (oxidised), h = lysozyme. The cytochrome C peaks were identified by oxidation with potassium ferricyanide and reduction with sodium dithionite [47]...

Prepare a fresh set of protein standards by diluting the 2.0 mg per ml BSA stock standard (stock solution) as shown in Table 14.2. There will be sufficient volume for three replications of each diluted BSA standard, if necessaiy. 

Figure 4-4. Use of SDS-PAGE to observe successive purification of a recombinant protein. The gel was stained with Coomassie blue. Shown are protein standards (lane S) of the indicated mass, crude cell extract (E), high-speed supernatant liquid (H), and the DEAE-Sepharose fraction (D). The recombinant protein has a mass of about 45 kDa.

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-... 

Figure 17 SDS-capillary gel electrophoresis of protein standards. Separation conditions 27 cm x 50 pm i.d. uncoated capillary 888 V/cm gel eCAP 200. (From Bene-dek, K. and Guttman, A., ]. Chromatogr., 680, 375, 1994. With permission.)...

FIGURE 6.6 Merck Chromolith monolithic RPLC column at 1 mL/min (top) and 2 mL/min with various injection volumes. Protein standards A = aprotinin, B = cytochrome C, C = carbonic anhydrase. 

This instrument was used to analyze mixtures of intact proteins, including protein standards and a cell lysate of the bacterium Escherichia coli. A UV... 

Size-based analysis by CE provides similar information and comparable limits of detection to analysis by SDS-PAGE with Coomassie blue staining.120 129 The performance of both electrophoretic techniques for the analysis of polypeptides is far superior to size exclusion chromatography. Figure 9.7 shows the separation of SDS-complexed recombinant protein standards by CE. 

Check transfer efficiency by staining the gel after transfer, or by staining a second blot with a total protein stain, such as coomassie blue or ponceau red. Alternatively, use commercially available prestained protein standards that are run along the samples of interest and that are visible during both the separation electrophoresis and on the membrane after transfer... 

Amino acids in proteins Standard abbreviation Molecular mass ... 

The abundance and ease of purification made bovine serum albumin (BSA) an early standard in protein chemistry, and BSA is widely used as protein standard in biuret, Lowry, and Bradford assays as well as a molecular weight... 

About 1 - 5 pi of the pre-mixed ready-to-use protein standards per lane are recommended in SDS-PAGE combined with Coomassie staining. 

MagicMark XP Western Protein Standard was used for estimation of the apparent molecular masses of the HaloTag-fusion proteins. 

After transfer, cut the membrane along the lane of prestained protein standards. Keep the piece of PVDF membrane with original, underivatized glycoproteins in a buffer for future elutions. Proceed with the detection of biotinylated glycoproteins. 

Pool all the fractions contained within the main antibody peak, 1 e, corresponding to an Mr of 150,000 relative to protein standards. Discard any flanking fractions that may contain aggregated protein or contaminants of lower molecular weight... 

The selection of a protein standard is potentially the greatest source of error in any protein assay. Of course, the best choice for a standard is a highly purified version of the predominate protein found in the samples. This is not always possible nor always necessary. In some cases, all that is needed is a rough estimate of the total protein concentration in the sample. For example, in the early stages of purifying a protein, identifying which fractions contain the most protein may be all that is required. If a highly purified version of the protein of interest is not available or it is too expensive to use as the standard, the alternative is to choose a protein that will produce a very similar color response curve with the selected protein assay method. 

Prepare a dilution series from protein standard (e.g., 2 mg/ml BSA) and sample buffer to cover the range 100 to 1000 pg/ml. 

Ideally, for purified or partially purified protein, the protein standard should have an aromatic amino acid content similar to that of the sample protein. For the total protein of a crude lysate, bovine serum albumin (BSA) is a commonly used standard for spectrophotometric quantitation of protein concentration. A 3 mg/ml solution of BSA should have an A280 of 1.98, based on an A2S0 of 6.61 for a 1% (w/v) solution. 

---