Silver standard curve

To quantitate proteins from staining, a densitometer aided by computer software is used to evaluate band areas of samples compared to band areas of a standard curve. Amido black, Coomassie Brilliant Blue, and silver stains are all appHcable for use in quantification of proteins. 

A series of silver standards gave a linear calibration curve in flame atomic absorption with a slope of 807 meter units per ppm Ag in the standard. (The meter units are arbitrary numbers proportional to absorbance, and ppm refers to ig Ag/mL.) An unknown cyanide solution passed through the silver membrane gave a meter reading of 198 units. Find the molarity of CN in the unknown-... 

Fig. 2.4. Vector characterization. (A). SDS acrylamide gel electrophoresis of a rAAV-UF-11 vector. Approximately 2.5 x 1011 drp were loaded on a 10% SDS-polyacrylamide gel. The three capsid protein bands in the correct stoichiometry are visualized by silver staining. (B) Infectious center assay of a rAAV-CB-hAAT stock when probed for the transgene each spot represents a vector infected cell. (C) Dot blot analysis of the rAAV stock to determine the titer of DNase-resistant particles of rAAV. Amounts (ng) refer to a serial dilution of the packaged rAAV vector plasmid used to construct the standard curve. The stock has a calculated titer of 4.3 x 1013 drp/ml.

It has been repeatedly reported that silver stained methods are not suitable for computerized quantitation because of their capriciousness and nonlinearity. This is apparently true of the stains based on reduction with citric acid and weak carbonate because there is no predicting the slope of a plot of integrated intensity versus protein concentration without the use of a reliable and reproducible color. Thus, in order to use quantitation with these two methods one must perform a standard curve with each protein as its own standard or accept some relative standard for normalization. The relative approach has been successfully used with GELCODE and allowed measurement of protein changes within an experimental protocol (1 ). These disadvantages have discouraged the acceptance of silver staining to its full potential application. 

Block (96) reported an analytical method for chloride-bromide mixtures utilizing DSC. The fact that the heat of fusion of an ideal solid solution of the type AWX — BmX or AmX — AWY is directly proportional to the concentration of solute ion was used to determine chloride-bromide mixtures in the concentration range 0-100%. Solutions containing both chloride and bromide are precipitated with silver nitrate, forming solid solutions of silver chloride-bromide. The heat of fusion of the mixed crystal is then determined, and the percent chloride or bromide obtained from a previously prepared standard curve. 

Figure 14.10. Variation in Ag Ka intensity with mineralogical composition. A = low-Z matrix = high-Z matrix. The line is a calibration curve derived from silver standards prepared in a silica matrix. From P. G. Burkhalter, Anal. Chem., 43, 10 (1971), by permission of the publisher. Copyright 1971 by the American Chemical Society.

A similar method with an accuracy of 5% utUizes the ammoniacal sUver nitrate reaction (176). The sugars are separated in a solvent composed of ethyl acetate/ pyridine/water (2.6 1.0 3.5 v/v) made 0.15 N with respect to silver nitrate. After separation the paper is air-dried for 1 hour, placed in an ammonia atmosphere for 1 hour, and then heated for 20 minutes at 80 1 °. The densities of the dark brown spots are measured by direct photometry, using a densitometer. Reference is then made to a standard curve prepared by plotting logarithms of concentrations of known solutions against the densities. 

Fig. 3. Temperature-time curve for standardizing silver-enhancement Silver-enhancement time in IGSS increases with lower operating temperature The use of a calibration curve assists in optimizing the enhancement procedure...

Eumstead ( ) chose native fluorite as an internal standard for the analysis of quartz in coal dust His procedure consisted of mixing 0 20 mg fluorite into each water suspension of standard and sample and depositing on silver membrane filters for a calibration curve (fluorite/quartz intensity ratio i[ . mg quartz) The application of this method to coal dust samples containing less than 1% quartz produced acceptable results relative standard deviation (RSD) was 18 2 ... 

In order to get a clearer picture of conditions existing on deposition of metals, let us discuss some examples. The deposition potential of silver from a normal solution of its salt almost equals the standard reduction potential tca = + 0.8 V, and the potential of hydrogen evolved from a neutral solution Ttn, equals 0.059 log 10-7 = —0.41 V. Both potentials are so wide apart that not even the polarization occurring at higher current densities can considerably affect the relative position of both curves. For this reason, silver will be deposited from the solution prior to hydrogen until practically all Ag+ ions will be... 

Figure 5. A plot of the amount of nuclease complex formed in the continuous variation titration experiments versus mole fraction of the R subunit as determined by densitometry of silver stained gels such as that shown in figure 4. The lines drawn are the theoretical curves expected for the association of 1 (. ..), 2 (-), or 3 (—) R subunits per molecule of MjS, mtase. The error bars are +/- one standard deviation.

Two x-ray fluorescence spectrometers were used for the analyses a General Electric XRD-6 for iron, copper, tin, and antimony, and a General Electric XRD-5 for nickel, silver, and lead (the latter machine has updated electronics and gave superior results for these three elements). Four certified standards from the National Bureau of Standards were used for each element to obtain a straight line calibration curve using linear regression (10). The experimental conditions used for the determination of each element were given by Carter et al. (10). 

The most common method of determining the halide ion concentration of aqueous solutions is titration v /ith a standard solution of silver nitrate. The reaction product is solid silver halide. A titration curve for this method usually consists of a plot of pAg versus the volume of silver nitrate added. To construct titration curves, three type of calculations are required, each of which corresponds to a distinct stage in the reaction (1) preequivalence, (2) equivalence, and (3) postequivalence. Example 13-10 demonstrates how pAg is determined for each of these stages. 

Fig. 23.10 Change of redox potential at an Ag/Ag electrode diving potentiometric titration of a silver-containing solution with a KCI standard solution (solid line) and first derivative of the titration curve (dotted line).

Take volumes of standard silver solution containing increasing quantities of Ag+ between 0.1 and 100 pg and subject each one to the entire process of analysis. Take the measurements at 462 mn. In the range up to 100 pg of Ag+/25 ml the calibration curve is slightly curved. 

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