Protein concentration measurement

As described in the following chapter, there are many biopharmaceutical applications of protein assays. Assigning the protein concentration for the drug substance, drug product, or in-process sample is often the first task for subsequent analytical procedures because assays for purity, potency, or identity require that the protein concentration be known. Hence it is typical for several different methods to be employed under the umbrella of protein concentration measurement, depending on the requirements of speed, selectivity, or throughput. The protein concentration is valuable as a stand-alone measurement for QC and stability of a protein. However, protein concentration methods provide no valuable... 

Figure 1. Elution patterns of the ammonium sulfate-precipitated enzyme preparation from a DEAE-Sephadex A-50 column. (0) CMC-saccharifying activity (5-min incubation) of eluates diluted 60-fold, (O) Avicel-saccharifying activity (1-hr incubation), ( ) protein concentration measured in terms of the absorbance at 280 nm column 5.0 X 50 cm flow rate 20 mL/8 min one fraction 20 mL.

The evaluations of the activities were done by extracting the necessary product concentration out of the chromatogram, calculating conversion of the enzymatic reaction, and using the intracellular protein concentration, measured by the method of Bradford [33]. 

Experimental errors tend to be quite large in biological systems, e.g. 30% in protein concentration measurements. Cell number measurements are generally no better than 5%. At lower viabilities (< 70% viable), accurate determination of viability and cell number is difficult, and the error in each determination may be greater than 10%. Errors in cell and metabolite concentration measurements lead to uncertainties in calculated parameters, such as specific growth, production and consumption rates, therefore a complex profile for these calculated parameters should not be assumed when a straight-line or simple function will suffice. 

The laboratory tests used most commonly to assess hypovolemia are packed cell volume (PCV) and plasma total solids. Unfortunately, these tests are neither sensitive nor specific (Hansen DeFrancesco 2002). The PCV may be increased substantially by splenic contraction, making small increases very hard to interpret. A PCV of >50% usually represents hypovolemia. The plasma total solids (protein measured by refractometer) or total protein concentration (measured by a chemistry analyzer) also increases with hypovolemia. How-ever significant protein loss can occur in disease (particularly colitis), resulting in a low or normal protein concentration despite hypovolemia. Further, hypergammaglobulinemia (e.g. in cyathostomiosis) can increase the plasma total protein concentration without the presence of hypovolemia. The PCV and plasma total solids are most useful when greatly increased or when used serially to monitor the response to fluid therapy. 

Moro, A., Gatti, C., and Delorenzi, N. (2001). Hydrophobicity of whey protein concentrates measured by fluorescence quenching and its relation with surface functional properties. J. Agric. Food Chem. 49, 4784-4789. 

Take samples from each step for acid phosphatase activity and protein concentration measurements (tee Note 8). 

To investigate the mechanism of the BSA on the improvement of enzymatic hydrolysis of pretreated CWR, the adsorptions of BSA on various substrates were studied [17]. Three sets of 20-ml vials were used to test the adsorption of BSA to Avicel, pretreated CWR, and lignaceous residue, respectively. The mixtures of citrate buffer (pH=4.8) with 8% (w/w) Avicel, 8% (w/w) pretreated CWR, or 3% (w/w) lignaceous residue were preheated to 50°C for 30 min. BSA was then added to the vials with the ratio of BSA to dry solid equal to 0.1 g BSA/g dry solid. The final working volume was 10 ml. One-milliliter aliquots were periodically withdrawn at start and after 1, 2, 4, 8, 24, 48, and 72 h. The aliquots were pretreated according to the procedures described in Effect of Tween 20 and BSA on Enzyme Protein Concentration and Activity for BSA protein concentration measurement. 

The final protein concentration should be in the range of 0.8-1.2 mg/mL, which is the minimum required for the C1 uptake experiments. Depending on the protein concentration measured initially, adjust the final volume to 200-300 pL CA 3 or 100-200 pL DG with HB. Keep the preparations in Eppendorf tubes on ice. 

Kit and equipment for spectrophotometric protein concentration measurement by Lowry, Bradford, or bicinchoninic acid (BCA) assay (Tliermo [San Jose, CA] or equivalent)... 

Rodriguez-Ruiz, 1., et al., 2015. A multiple path photonic lab on a chip for parallel protein concentration measurements. Lab Chip 15 (4), 1133-1139. Available at http //xlink.rsc. org/ DOl=C4LC01332H. 

Protein concentration can be determined by using method of Bradford,9 which utilises Pierce reagent 23200 (Pierce Chemical Company, Rockford, IL, USA) in combination with an acidic Coomassie Brilliant Blue G-20 solution to absorb at 595 nm when reagent binds to the protein. A 20 mg/1 bovine serum albumin (Pierce Chemical) solution was used as the standard. Starch concentration was measured by the orcinol method4,9-11 using synthetic starch as the reference. A yellow to orange colour is obtained and measured at 420 nm when orcinol reacts with carbohydrates. Absorbance is determined by spectrometry. 

Eleven controlled diet and environment experiments have been designed in a way that can be used to investigate the effects of protein nutrition and heat and/or water stress on diet-tissue A N. Laboratory rats were raised on purified, pelletized diets in which the isotopic composition of proteins, lipids and carbohydrates were well characterized and their proportions accurately and precisely measured (Ambrose and Norr 1993). Four experiments involved manipulation of temperature and/or water availability. Of these four experiments, one used a diet with high (70%) protein concentrations and heat/water stress (36°C) and three used normal (20%) protein concentrations. Seven experiments were conducted at normal temperature (21°C) with water ad libitum. Of these seven experiments, two used diets formulated with veiy low protein (5%), three with normal protein and two with high protein concentrations. 

The rejection coefficient R) was calculated according to the following equation / = In (Cr/C )/ln (VJVr). Cr or Vr represent the protein concentration in the retentate or the volume of the retentate Co is the concentration of the protein in the solution before filtration 1 is the initial volume of the feed. The pH value of each protein solution was immediately measured after dissolving the proteins in distilled water. 

Assays. Protein concentrations were measured by the method of Bradford (18) and the various contractile protein ATPase activities by tRe method of Martin and Doty (19). Gel electrophoresis was carried out by the method of Ames (20) on 1.5 ran polyacrylamide slabs using the discontinuous SDS buffer system of Laemnli (21). Dried gels were scanned at 550 nm for densiometry measurements. 

For acute symptomatic hypocalcemia, 200 to 300 mg of elemental calcium is administered IV and repeated until symptoms are fully controlled. This is achieved by infusing 1 g of calcium chloride or 2 to 3 grams of calcium at a rate no faster than 30 to 60 mg of elemental calcium per minute. More rapid administration is associated with hypotension, bradycardia, or cardiac asystole. Total calcium concentration is commonly monitored in critically ill patients. Under normal circumstances, about half of calcium is loosely bound to serum proteins while the other half is free. Total calcium concentration measures bound and free calcium. Ionized calcium measures free calcium only. Under usual circumstances, a normal calcium level implies a normal free ionized calcium level. Ionized calcium should be obtained in patients with comorbid conditions that would lead to inconsistency between total calcium and free serum calcium (abnormal albumin, protein, or immunoglobulin concentrations). For chronic asymptomatic hypocalcemia, oral calcium supplements are given at doses of 2 to 4 g/day of elemental calcium. Many patients with calcium deficiency have concurrent vitamin D deficiency that must also be corrected in order to restore calcium homeostasis.2,37,38... 

Detection and quantification of protein by measuring absorbency at 280 nm is perhaps the simplest such method. This approach is based on the fact that the side chains of the amino acids tyrosine and tryptophan absorb at this wavelength. The method is popular, as it is fast, easy to perform and is non-destructive to the sample. However, it is a relatively insensitive technique, and identical concentrations of different proteins will yield different absorbance values if their content of tyrosine and tryptophan vary to any significant extent. Hence, this method is rarely used to determine the protein concentration of the final product, but it is routinely used during downstream processing to detect protein elution off chromatographic columns, and hence track the purification process. 

The most common methods used to determine protein concentration are the dye-binding procedure using Coomassie brilliant blue, and the bicinchonic-acid-based procedure. Various dyes are known to bind quantitatively to proteins, resulting in an alteration of the characteristic absorption spectrum of the dye. Coomassie brilliant blue G-250, for example, becomes protonated when dissolved in phosphoric acid, and has an absorbance maximum at 450 nm. Binding of the dye to a protein (via ionic interactions) results in a shift in the dye s absorbance spectrum, with a new major peak (at 595 nm) being observed. Quantification of proteins in this case can thus be undertaken by measuring absorbance at 595 nm. The method is sensitive, easy and rapid to undertake. Also, it exhibits little quantitative variation between different proteins. 

Analysis of CSF chemistries typically includes measurement of glucose and total protein concentrations. An elevated CSF protein of 100 mg/dL or greater... 

There are two principle ways for optical detection of protein concentrations either the macromolecule or its label emits energy (after excitation by light) -then a fluorescence signal can be measured or it absorbs energy from electromagnetic waves passing the sample - then the optical absorption of the sample can be measured by UV/Vis spectroscopy and concentrations can be calculated according to Lambert-Beers Law. 

Kennedy, S.W. and Jones, S.P. 1994. Simultaneous measurement of cytochrome P4501A catalytic activity and total protein concentration with a fluorescence plate reader. Anal. Biochem. 222 217. 

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