Measurement of Water Activity by Electronic Sensors
Water activity of foods is an important thermodynamic property affecting stability with respect to physical, chemical and microbiological changes. Water activity, aw, is the ratio of the vapor pressure of water in a system, Pw sy, to the vapor pressure of pure water, Pv w, at the same temperature. It is equal to the equilibrium relative humidity ERH established in the surrounding air. Thus Several instruments based on electronic sensors are commercially available for direct determination of...
Nitrogen Determination Using The Dumas Method
Nitrogen, freed by pyrolysis and subsequent combustion, is swept by a carbon dioxide carrier into a nitrometer. The carbon dioxide is absorbed in KOH and the volume of residual nitrogen is measured and converted to equivalent protein by a numerical factor AOAC, 1999b . CuO-Pt catalyst CuO wire form with 2.5 Pt reforming catalyst Laboratory mill No. 30 sieve Bottles with caps Nitrogen analyzer, consisting of combustion and collection and measuring systems e.g., PE Biosystems Vycor combustion...
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occurrence , a periodicity of appearance, and secondary structure parameters. Kato and Nakai 1980 found that the effective hydrophobicity measured by using cis-parinaric acid CPA see Basic Protocol 1 could explain surface properties of proteins that were not necessarily directly correlated with the average hydrophobicity. Since then, the importance of surface hydrophobicity in protein functions has been widely recognized. As a recent example, the effect of oxygen radical-dependent oxidation...
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at hand. Any method should be tested before use to make sure results will correlate with the real food system under study. Mangino 1989 stated that the method of functional property evaluation many have a greater effect on the results than the actual variables under study. Some methods are highly standardized procedures, whereas others are specific to a particular laboratory. Some methods are highly empirical, others are more fundamental. No single test will work for all applications. Many...
Microtiter Plate Modified Lowry Assay
The modified Lowry assay can also be done in a 96-well microtiter plate format. The assay has a working range of 1 to 1500 g ml. Additional Materials also see Alternate Protocol 1 Microtiter plate and cover or tape seals 200- l multichannel pipettor Microtiter plate reader for 750 nm 1. Draw a template for placement of samples and standards on the microtiter plate. 2. Add 40 l of each diluted BSA standard see Alternate Protocol 1, step 1 , sample, or diluent blank to the appropriate wells of a...
Probe Spectrofluorometry Using Dph
DPH has been used to eliminate the charge effects observed when using ANS Tsutsui et al., 1986 . Because the dissociation constant of ANS is on the order of 10-3 to 10-4 equivalent to relatively strong organic acids such as butyric and caproic acids , it is highly soluble in water and pH changes have only a slight effect on the quantum yield. CPA is even better in this regard. Nevertheless, some charge effects cannot be ignored due to the presence of a very weak carboxylic charge on the...
Basic Theory And Interpretation Of Fluorescence Spectra
When radiation is incident on a solution, most of it is scattered without change in wavelength. Thus, exciting radiation at 280 nm will be scattered and a fraction of it will be detected by the spectrofluorometer as a peak at the same wavelength. The width of this Rayleigh scattering peak Fig. B3.6.1 will depend on instrumental settings, mainly those of the excitation and emission slit widths, which determine the range of wavelengths observed. The bandwidth is the width, in nm, of the...
Microtiter Plate Assay For Bca Measurement Of Total Protein
BCA assays can be run in 96-well microtiter plates. The assay has a working range of 125 to 2000 g ml. Additional Materials also see Alternate Protocol 3 96-well microtiter plate with cover or tape seal 200- l multichannel pipettor Microtiter plate shaker 37 C dry-heat incubator Microtiter plate reader 1. Draw a template for planning samples and standards on a microtiter plate. Blanks, standards, and samples should be prepared in triplicate. 2. Add 10 l of each diluted BSA standard see...
The Colorimetric Detection and Quantitation of Total Protein
Protein quantification is an important step for handling protein samples for isolation and characterization, and is a prerequisite step before submitting proteins for chromatographic, electrophoretic, or immunochemical analysis and separation. The methods included in this unit are colorimetric measurements, whose procedures are faster, simpler, and less laborious than those based on estimation of total nitrogen content unit B1.2 . This unit describes four of the most commonly used total protein...
Protein Digestibilitycorrected Amino Acid Score Pdcaas To Determine Protein
The protein digestibility-corrected amino acid score PDCAAS has been adopted as a simpler approach for determining protein quality on a routine basis. No animal feeding studies are required. An additional advantage is that the method can often more closely predict the protein quality of a protein ingredient for humans than can assays in the growing rat. It is the method of choice for highly digestible proteins that contain minimal amounts of antinutritional factors. PDCAAS is calculated as the...
Literature Cited Fel
Campbell, G.S. and Lewis, D.P. October 1998. Water activity and dew point temperature measuring apparatus and method. U.S. patent 5,816,704. Chirife, J. and Resnik, S.L. 1984. Unsaturated solutions of sodium chloride as reference sources of water activity at various temperatures. J. Food Sci. 49 1486-1488. Fontana, A.J. 1998. Water activity Why it is important for food safety. In Proceedings of the First NSF International Conference on Food Safety, Albuquerque, N. Mex., pp. 177-185. Greenspan,...
Coomassie Blue R250 Staining
Coomassie blue R-250 can be used with most types of blot membranes except nitrocellulose high concentrations of organic solvents can dissolve nitrocellulose membranes . Coomassie blue has a similar sensitivity to amido black. Coomassie blue-stained proteins gt 50 ng band appear as dark blue bands against a light blue background. The sequence of washing with water, staining, and destaining is similar to that for amido black staining Basic Protocol 1 , but the staining step is lengthened to 5...
Nitrogen Determination Using The Kjeldahl Method
Since late in the 19th century, the classical Kjeldahl method has been recognized and accepted universally as the authoritative method of analysis for determining the protein content in a wide variety of ingredients and finished products. As a result of technical innovations there are currently available and in use semiautomated or fully automated protein analysis systems that are based on the classical Kjeldahl procedure. The Kjeldahl method determines total nitrogen content and protein as the...
Native Isoelectric Focusing
Isoelectric focusing IEF separates proteins according to their isoelectric point pI . The technique is performed in thin polyacrylamide gels incorporating carrier ampholytes, which are small amphoteric compounds that form a pH gradient when voltage is applied across the gel. Proteins introduced into this pH gradient will migrate until they reach their isoelectric point. Each protein species in the sample can be visualized as a sharply focused band. IEF is best performed in a flatbed...
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means that a greater degree of purification is needed, the fact that membrane proteins have, by definition, properties somewhat different from those of cytoplasmic proteins permits some very effective purification steps e.g., hydrophobic chromatography or fractional solubility separation . Peripheral membrane proteins are only loosely attached and may be released by gentle conditions such as high pH, EDTA, or low nonionic detergent concentrations. Once in solution, some peripheral proteins no...
Ninhydrin reagent
Dissolve 0.8 g SnCl22H2O in 500 ml 200 mM citrate buffer see recipe to give a 7 mM SnCl2 solution. Dissolve 20 g crystalline ninhydrin in 500 ml 2-methoxyethanol methyl cellosolve, Brenntag to give a 4 w v solution. Combine 500 ml SnCl2 solution and 500 ml ninhydrin solution. Purge with nitrogen and store in dark bottles for up to one week at 4 C. CAUTION Methyl cellosolve is toxic and ninhydrin stains everything it contacts. Biochemical Compositional Analyses of Proteins Evaluation of the...
Protein Elution From Pvdf Membranes Using Acidic Extraction With Organic
Blotted proteins are usually of high purity and thus good candidates for additional characterization. If the protein cannot be further characterized on the blot and contamination of samples with large amounts of detergent Basic Protocol 2 is unacceptable, acidic elution with organic solvents may be an alternative. The use of organic solvents limits this protocol to PVDF membranes, which have high chemical resistance. This protocol may produce highly variable recoveries for different proteins...
Glutaraldehyde fixing solution
4 ml 25 glutaraldehyde final 0.2 150 ml ethanol final 30 13.61 g sodium acetate trihydrate final 0.2 M Lower anodic Tris-tricine tank buffer Dissolve 121.1 g Tris base final 0.2 M and 10 g SDS final 0.1 in 500 ml water. Adjust pH to 8.9 with HCl. Dilute to 5 liters. Store lt 1 month at room temperature. 1.6 ml 4x stacking gel buffer see recipe Store 0.5-ml aliquots lt 6 months at -20 C Dissolve 36.3 g Tris base final 1.5 M in 150 ml water. Adjust pH to 8.8 with HCl. Bring volume to 200 ml with...
Ponceau S Staining
Ponceau S is the least sensitive general protein stain described here. Transferred proteins gt 200 ng band appear as red bands on a pink background. Major advantages of Ponceau S staining are that it is simple, rapid, and reversible. If desired, essentially all of the stain can be removed by extended destaining as described in steps 4 to 6. This can be particularly advantageous if the blot is to be reused after initial protein staining for a second detection method such as immunoblotting.
Native Discontinuous Page
Under native conditions, polypeptides retain their higher-order structure and often retain enzymatic activity and interactions with other polypeptides. The migration of proteins under native conditions depends on many factors including size, shape, and native charge. One straightforward approach to native electrophoresis is to leave out the SDS and reducing agent DTT or 2-mercaptoethanol from the SDS-PAGE protocol see Basic Protocol 1 . Prepare all solutions without SDS, including tank buffer....
Key References Jlg
Contains tables of the water activity of saturated salt solutions at various temperatures. Rockland, L.B. and Beuchat, L.R. eds. 1987. Water Activity Theory and Application to Food. Marcel Dekker, New York. Describes in detail the mechanisms of water-food interactions. Rockland, L.B. and Stewart, G.F. eds. 1981. Water Activity Influences on Food Quality. Academic Press, New York. Describes thephysicochemical and biological influences of water activity on food. Describes the role of water...
Modified Lowry Protein Assay For Determination Of Total Proteins
Preformulated, stabilized, modified versions of the Lowry reagent are now commercially available from Pierce the Modified Lowry Protein Assay Reagent or from Bio-Rad the DC Protein Assay . The assay can be performed in test tubes or a microtiter plate see Alternate Protocol 2 . The working range of this assay is 1 to 1500 g ml if the Pierce reagent is used, or 200 to 1400 g ml if the Bio-Rad reagent is used. Table B1.1.1 is a brief troubleshooting guide for this technique. Additional Materials...
Quenching
Fluorescence quenching provides an indicator of the solvent accessibility of tryptophans and of the dynamics of protein conformation. The presence of certain extrinsic groups in physical contact with an excited chromophore can lead to sharing or transfer of the excitation energy, with consequent reduction in the quantum yield and diminution of fluorescence intensity. The efficiency of this quenching depends on the electronic structure of the extrinsic molecule or ion. Because it also depends on...
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Measuring absorbance at 280 nm A280 is one of the oldest methods for determining protein concentration Warburg and Christian, 1942 Layne, 1957 . This method is still widely used because it is simple and does not require incubating the sample with exogenous chromo-phores. However, the detection limit is higher than colorimetric methods and therefore higher concentrations of protein are necessary. The A280 method requires that the protein being quantitated have aromatic amino acids, primarily...
Determining the CD Spectrum of a Protein
Many of the complex phenomena associated with food processing, including cooking, involve the interactions of biopolymers. These interactions are frequently triggered by changes in the three-dimensional conformation of the biopolymers, which may be caused by changes in temperature, pH, or ionic strength, or by modifications made by hydrolytic enzymes. The understanding of such processes, necessary for their control, requires knowledge of the dependence of the conformational changes and...
The Lowry Protein Assay For Determination Of Total Proteins
In 1951, Oliver H. Lowry introduced this colorimetric total protein assay method. It offered a significant improvement over previous protein assays, and his paper became one of the most cited references in the life-science literature Lowry, 1951 . The Lowry assay is easy to perform, since the incubations are done at room temperature and the assay is sensitive enough to allow the detection of total protein in the low microgram per milliliter range. It is one of three copper chelation...
The Coomassie Plus Protein Assay For Determination Of Total Protein
Several companies offer modified Bradford Coomassie dye-based protein assay reagents. Perhaps the most popular such reagent is the Protein Assay Reagent available from Bio-Rad. The Coomassie Plus Protein Assay Reagent available from Pierce is another modification of the Bradford formulation. In addition to the attributes cited above, the Coomassie Plus Protein Assay Reagent has the unique advantage of producing a linear response curve within a portion of its working range. For B SA, the...
Background Information Seo
Electrophoresis is the process of moving charged molecules in solution by applying an electrical field across the mixture. Because molecules in an electrical field move with a speed dependent on their charge, shape, and size, electrophoresis has been extensively developed for molecular separations. As an analytical tool, electrophoresis is simple, relatively rapid, and has unparalleled resolving power. It is used chiefly for analysis and purification of very large molecules such as proteins and...
Measurement of Water Activity Using Isopiestic Method
Water is an important constituent of all foods. In the middle of the twentieth century, scientists began to discover the existence of a relationship between the water contained in a food and its relative tendency to spoil. They also began to realize that the chemical potential of water is related to its vapor pressure relative to that of pure water i.e., the relative vapor pressure RVP . This RVP was termed water activity or aw. Scott 1957 clearly stated that the aw of a medium correlated with...
Secondary Antibodyprotocol
The following procedure is based on the Vectastain ABC kit from Vector suppliers appendix . It uses an avidin-biotin complex to attach horseradish peroxidase HRP or alkaline phosphatase AP to the biotinylated secondary antibody. Avidin-biotin systems are capable of extremely high sensitivity because multiple reporter enzymes are bound to each secondary antibody. In addition, the detergent Tween 20 is a popular alternative to protein blocking agents when using nitrocellulose or PVDF membranes....
Using 280 To Determine Protein Concentration
Determination of protein concentration by measuring absorbance at 280 nm A280 is based on the absorbance of UV light by the aromatic amino acids tryptophan and tyrosine, and by cystine, disulfide bonded cysteine residues, in protein solutions. The measured absorbance of a protein sample solution is used to calculate the concentration either from its published absorptivity at 280 nm a280 or by comparison with a calibration curve prepared from measurements with standard protein solutions. This...
Linear Gradient Page
Gradient gels, although more difficult to cast than single-concentration gels, fractionate a wider size range of proteins on a single gel. Furthermore, calculating molecular weights see Support Protocol 3 is simplified because, unlike single-concentration gels, the relationship between the logarithm of the molecular weight of a protein and its mobility is linear over most of the fractionation range of a gradient gel. A gradient maker mixes a high- and a low-percentage acrylamide solution during...
Using Fluorescence Emission To Determine Protein Concentration
Protein concentration can also be determined by measuring the intrinsic fluorescence based on fluorescence emission by the aromatic amino acids tryptophan, tyrosine, and or phenylalanine. Usually tryptophan fluorescence is measured. The fluorescence intensity of the protein sample solution is measured and the concentration is calculated from a calibration curve based on the fluorescence emission of standard solutions prepared from the purified protein. This assay can be used to quantitate...
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Dioxetane phosphate substrate buffer 1 mM MgCl2 0.1 M diethanolamine 0.02 w v sodium azide optional Adjust to pH 10 with HCl and use fresh Traditionally, the AMPPD substrate buffer has been a solution containing 1 mM MgCl2 and 50 mM sodium carbonate bicarbonate, pH 9.6 Gillespie and Hudspeth, 1991 . The use of diethanolamine results in better light output Western Light instructions Applied Biosystems . Alternatively, 100 mM Tris-Cl pH 9.5 100 mMNaCl 5 mMMgCl2 can be used Sandhu et al., 1991 ....
Activity Staining For Proteinase
Proteinases in complex protein samples are separated and detected by their activity using a technique called substrate-SDS-PAGE. After electrophoresis, the gel is incubated in a casein substrate solution and then stained with Coomassie brilliant blue. The presence of active proteinase is indicated by clear zones on a blue background. Information about the number of active components and their molecular weights is obtained by this technique. This method is useful when studying the presence,...
Net Protein Utilization Npu And Biological Value Bv To Determine Protein Quality
An NPU assay can be conducted in conjunction with a PER assay. NPU is used as a means of predicting the biological value of a protein. For NPU assays, the protein content of the animal carcasses is determined. Biological value BV measures the efficiency of the utilization of nitrogen by the animal. The biological value of a test protein is the proportion of nitrogen that is retained for maintenance and for growth, corrected for metabolic and endogeneous losses of nitrogen BV N retained N...
In Vitro Protein Digestibility For Cper Aoac Method 43265
C-PER is a PER calculated from the amino acid composition of the test protein and an in vitro protein digestibility measurement. Its calculation involves complicated algorithms provided as part of AOAC procedures. Additional Materials also see Basic Protocol 3 Bacterial protease Pronase P or E 55 C water bath 1. Prepare these two enzyme solutions as per AOAC 43.261 Solution A Make up fresh and store on ice. Dissolve 227,040 BAEE units of trypsin and 1860 BAEE units of a-chymotrypsin, 0.520...
Direct Manometric Determination of Vapor unit a24 Pressure
Water activity aw is the ratio of the partial vapor pressure of water above a solution to that of pure water at the same specific temperature. It plays an important role in evaluating the microbial, chemical, and physical stability of foods during storage and processing. The vapor pressure in the headspace of a food sample can be measured directly by a manometer. A manometer has one or two transparent tubes and two liquid surfaces where pressure applied to the surface of one tube causes an...
Sdspage In A Tristricine Buffer System
The Tris-glycine discontinuous buffer system of Laemmli cannot be used for the separation of proteins with molecular weights lt 10 to 15 kDa. For the analysis of smaller proteins, an alternative Tris-tricine buffer system is used along with an acrylamide solution that has a high percentage of cross-linker. This technique should be used when separating peptides in the size range of 1 to 20 kDa. Additional Materials also see Basic Protocol 1 Ethylene glycol 40 w v acrylamide solution see recipe...
Nitrogen Determination Using The Combustion Method
This is an instrument method derived from the Dumas method as an alternative method to the mercury catalyst Kjeldahl method. It has two advantages 1 less time is needed for nitrogen determination, and 2 hazardous and toxic chemicals are not utilized. Nitrogen freed by combustion at high temperature in pure oxygen is measured by thermal conductivity detection and converted to equivalent protein by an appropriate numerical factor AOAC, 1999 . This method is applicable to all flours, cereal...
Critical Parameters and Troubleshooting 1
Like the gravimetric methods, the Karl Fischer titration method requires only a very small amount of sample between 1 and 10 g depending on the moisture level . Therefore, sample homogeneity is very critical and special care must be given to sample blending before sub-sampling for analysis. The method used for homogenization will depend on the type of sample being analyzed. Blenders, homogeniz-ers, mincers, food processors, and graters are currently available for dry, moist, and very wet...
Chromogenic visualization solutions
BCIPNBTvisualization solution Mix 33 l NBT stock 100 mg NBT in 2 ml 70 v v dimethylformamide DMF , stored lt 1 year at 4 C and 5 ml alkaline phosphate substrate buffer see recipe . Add 17 l BCIP stock 100 mg BCIP in 2 ml 100 DMF, stored lt 1 year at 4 C and mix. Stable 1 hr at room temperature. Recipe is from Harlow and Lane 1988 . Alternatively, BCIP NBT substrates may be purchased from Sigma, Kirkegaard amp Perry, and Vector suppliers appendix . 4CN visualization solution Mix 20 ml ice-cold...
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This method uses the same materials and apparatus as described above see Basic Protocol 1 . In addition a standard material, such as microcrystalline cellulose MCC , starch, filter paper, glycerol, or polyethylene glycol is needed for this protocol. The standard material is selected based on the commercial availability of a material with high purity and consistency over time, moreover, the standard must be stable during reuse of the material. Thus, MCC is recommended Spiess and Wolf, 1987 ....
Amido Black Staining
Amido black is used to stain proteins on blot transfer membranes. Transferred proteins gt 50 ng band appear as dark blue bands on a light blue background. Amido black has a sensitivity similar to that of Coomassie blue, but it stains faster. It is the preferred stain for protein sequencing and in situ cleavage of proteins for determining internal sequences because the mild staining and destaining conditions minimize the likelihood that any protein will be extracted during the procedures.
Silver Staining Of Polyacrylamide Gels
Silver staining is approximately 50-fold more sensitive than Coomassie brilliant blue staining see Support Protocol 1 . It is a complex, multistep process, and many variables can influence the results. High-purity reagents and precise timing are essential for reproducible, high-quality results. Impurities in the gel and or the water used for preparing the staining reagents can give poor staining results. The detection limit of this technique is from 1 to 5 ng protein per band. This protocol...
Estimation Of Protein Molecular Weights By Sdspage
Estimating the molecular weight of a protein is relatively straightforward with SDS-PAGE. Molecular weight standards are separated on a polyacrylamide gel along with the unknown or sample protein, and the migration distance of each is measured. This distance is converted to relative mobility Rf , which is defined as the distance migrated by a protein divided by the distance migrated by a relative mobility marker. Usually the dye front is chosen as the relative mobility marker. Alternatively, a...
Iaedans Labeling
N-iodoacetyl-N- 5-sulfo- 1-naphthyl ethylenediamine IAEDANS or 1,5-I-AEDANS is used for fluorescent labeling of electroblotted proteins on blot transfer membranes. Because IAEDANS reacts with free cysteines, disulfides in the sample must first be reduced with dithiothreitol DTT . Transferred protein bands are visualized under UV light. Additional Materials also see Basic Protocol 7 DTT solution 200 mM dithiothreitol DTT in 100 mM TrisCl, pH 8.6 appendix2a prepare immediately before use 100 mM...
Fluorescamine Reaction To Determine Degree Of Hydrolysis
Fluorescamine reacts with primary amines to form fluorophores see Fig. B2.2.4 that are excited at 390 nm and fluoresce at 475 nm. Peptides react with fluorescamine at pH 7.0, giving higher fluorescence than amino acids, which have maximum fluorescence at pH 9. The reaction proceeds rapidly with primary amines at 25 C. The resulting fluorescence is proportional to the amine concentration. The fluorophores are stable for several hours. A negligible interference is produced with ammonia. Figure...
The Biuret Assay For Determining Total Protein
All proteins are composed of amino acids joined by peptide bonds in a linear sequence. There are 20 naturally occurring amino acids found in proteins. The amino acids are joined to each other by peptide bonds formed by a condensation reaction that occurs between the terminal amine of one amino acid and the carboxyl end of the next. Peptides containing three or more amino acid residues will form a colored chelate complex with cupric ions in an alkaline environment containing sodium potassium...
Formol Titration To Determine Degree Of Hydrolysis
Amino acids react with formaldehyde, liberating one H ion from the amino group, which is potentiometrically titrated with a sodium hydroxide solution. Formol titration is also useful for measuring total free amino acids for titration of acid tastants, see unit G2.1. Amino groups of histidine do not react and those of proline and hydroxyproline react about 75 . Tertiary amines such as those in the guanidine group do not interfere with the reaction. Protein containing significant amount of Pro...