Determination of microgram quantities of protein in 65-80 percent salt solutions

Due to the necessity of determining microgram quantities of protein in 65–80% salt solutions, a combination of gel filtration and low-ultraviolet absorption of proteins was investigated as a possible solution to this problem. Such a system is described and includes a spectrophotometer equipped with a strip-chart recorder to continuously monitor a Sephadex G-25 column. Also the construction of a flow meter, and the proper method of applying samples to the column when they are in high-salt solutions is described. A wavelength of 220 nm was selected over lower wavelengths because of greater light intensity and less stray light. At this wavelength the absorption of proteins seems to be less sensitive to changes in the composition of one amino acid, such as tryptophan, and more closely related to the total content of aromatic residues and the number of peptide bonds. Excellent results were obtained when the areas of the eluting protein peaks, but not the peak heights, were used to quantitatively measure 7–50 μg of protein. Although this method cannot be easily adapted for routine analysis of very large numbers of samples, it has the advantage of being more sensitive than the Lowry method and allows for the accurate determination of microgram quantities of protein in solvent systems that would preclude the use of other methods.     

Thin layer chromatography of commercial samples of amido black 10B

The tannic acid-phosphomolybdic acid-amido black (TPA) stain has been used primarily for staining hemoglobin. That different dye lots of amido black cause variable staining is documented in the literature. Nine commercial samples of amido black were investigated using thin layer chromatography; all of these dyes contained colored contaminants. Separation of contaminants was achieved using silica gel thin layer chromatography and a solvent system of 95% ethanol:90% phenol:concentrated NH4OH, 12:9:3. TPA staining of red blood cells was improved by using purified amido black.     

Some quantitative aspects of the disc electrophoresis of ovalbumin using amido black 10B stain

The quantitative aspects of the disc electrophoretic technique were investigated using a purified protein, egg ovalbumin. Depending on the filter used, a linear relationship between peak area and protein concentration was found up to about 40 μg of protein by densitometry. Both diffusion and gel slicing studies indicated that linearity could be extended to almost 160 μg of protein. By elution of the amido black dye from the protein-dye complex in the gel, a nearly constant dye to protein ratio was indicated. These results suggested that quantitation of the stained bands on polyacrylamide gels was limited by the nonlinear response of the densitometer, perhaps due to the nonlinearity of dye absorbance at large optical densities and not by variable amounts of dye binding to the protein bands.     

Comparison of digestion protocols for microgram quantities of enriched protein samples

Standard biochemical techniques that are used for protein enrichments, such as affinity isolation and density gradient centrifugation, frequently yield high-nanogram to low-microgram quantities at a significant expenditure of resources and time. The characterization of selected protein enrichments by the "shotgun" mass spectrometry approach is often compromised by the lack of effective and efficient in-solution proteolysis protocols specifically tailored for these small quantities of proteins. This study compares the results of five different digestion protocols that were applied to 2.5 mug portions of protein isolates from two disparate sources: Rhodopseudomonas palustris 70S ribosomal proteins, and Bos taurus microtubule-associated proteins (MAPs). Proteolytic peptides produced according to each protocol in each type of protein isolate were analyzed by one-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). The effectiveness of each digestion protocol was assessed on the basis of three parameters: number of peptide identifications, number of protein identifications, and sequence coverage. The two protocols using a solvent containing 80% acetonitrile (CH3CN) for trypsin digestions performed as well as, and in some instances better than, protocols employing other solvents and chaotropes in both types of protein isolates. A primary advantage of the 80% CH3CN protocol is that it requires fewer sample manipulation steps.     

Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system

We developed a cell-free protein synthesis system that produces more than 1mg/ml of recombinant proteins in two hours. A basal system that supports the stable maintenance of ATP and amino acids was constructed by using high concentrations of CP (100 mM) and amino acids (3 mM). Approximately 0.6 mg/ml of protein was produced during the batch incubation of the basal system. We found that the accumulation of inorganic phosphate reduces the concentration of free magnesium ions and that there exists a critical concentration of magnesium at which the protein synthesis is halted. Based on this finding, we attempted to extend the duration of the protein synthesis by keeping the magnesium concentration sufficiently high throughout the reaction period. The protein synthesis reaction continued for at least 2 h when the reaction was repeatedly supplemented with magnesium, and approximately 1.2 mg/ml of active CAT or GFP was produced. The simple, fast, and highly productive cell-free protein synthesis system described herein should offer a versatile platform for the preparation of protein molecules in various post-genomic efforts.     

Measurement of microgram quantities of protein by a generally applicable turbidimetric procedure

A modified turbidimetric method for protein determination which involves the use of trichloroacetic acid as the precipitating agent is described. Maximal turbidity develops in less than 30 min and is stable for at least 120 min. A linear relationship between turbidity at 340 nm and protein concentration is observed between 2 and 40 micrograms protein. Sodium dodecyl sulfate is added to avoid the interference by nonionic and cationic detergents and lipids and to decrease the protein-to-protein variation. The use of cetyltrimethyl ammonium bromide provides a two-step procedure to correct for the contribution of contaminating nucleic acid. Many compounds which interfere with other protein quantitation methods have no effect on this system. The interference of commonly used reagents as sucrose and urea can be easily corrected. This procedure compared favorably with the most widely used protein quantitation methods in simplicity, sensitivity, and specificity.     

Stoichiometry of the amido black reaction with proteins

Amido black 10B (C₂₂H₁₄N₆O₉S₂Na₂) reacted with nine different proteins to yield nearly identical molar ratios of sorbed dye to protein-bound basic amino acids.