Fetal bovine serum-a cell culture dilemma.

Ethical and possible reproducibility issues arise when using fetal bovine serum in cell culture media.

A Simple Whole-Blood Polymerase Chain Reaction without DNA Extraction for Thalassemia Diagnosis.

Polymerase chain reaction (PCR) diagnosis of thalassemia usually relies on using genomic DNA. Preparing the genomic DNA can lead to sample-to-sample contamination. This report was aimed to establish the PCR protocol using whole-blood for detecting mutations of α- and ß-globin genes causing the thalassemia syndrome. First, the PCR facilitators, betaine and bovine serum albumin (BSA), were tested, simultaneously with an adjustment of PCR thermal cycler and of whole-blood volume. Thereafter, the established whole-blood PCR was applied for detecting, in both known and unknown samples, the HBA1 Southeast Asian (- -SEA) (NG_000006.1: g.26264_45564del19301) deletion, Hb Constant Spring (Hb CS, HBA2: c.427T>C, αCSα), codon 17 (A>T) (HBB: c.52A>T), codons 41/42 (-TTCT) (HBB: c.126_129delCTTT) deletion, -28 (A>G) (HBB: c.-78A>G) and codon 26 (G>A) (Hb E or HBB: c.79G>A). It was shown that the whole-blood PCR worked successfully in 9.0% (w/v) betaine, with 1 µL of EDTA whole blood and with addition of 10 heat-cool steps (3 min. at 94 °C, followed by 3 min. at 55 °C) prior to the typical thermal cycles for the mutations. The capability of the new whole-blood PCR was similar to that of the typical DNA-based PCR. Therefore, the newly established whole-blood PCR could be performed for PCR diagnosis of thalassemia. Using this platform, sample-to-sample contamination should be eliminated.

Label-Free, Direct Measurement of Protein Concentrations in Turbid Solutions with a UV-Visible Integrating Cavity Absorbance Spectrometer.

Protein-particle conjugates and mixtures have been investigated extensively for their diverse applications in biotechnology. However, general methods to measure protein concentration of protein-particle solutions are lacking. Typically, proteins in turbid solutions require separation or staining with another chromophore to quantitate their concentration. Here we demonstrate a label-free, direct approach to measure protein concentrations in turbid solutions using a UV-vis integrating cavity absorbance spectrometer. Three systems are used to test the ability to measure accurate protein concentrations: proteins adsorbed to Alhydrogel, proteins in solution with gold nanoparticles, and proteins encapsulated within polymeric microspheres. Protein concentrations in each of the three protein-particle systems were successfully quantified using a calibration curve created from the absorbance at 280 nm.

Multicommutated flow analysis system for determination of total protein in cerebrospinal fluid.

A fully mechanized, computer-controlled, multicommutated flow analysis (MCFA) system dedicated for total protein determination in cerebrospinal fluid samples has been developed. For the protein determination the Exton method has been applied. Dedicated turbidimetric and nephelometric flow-through detectors operating according to paired-emitter detector diode principle have been fabricated by integration of two or three respective light emitting diodes. The developed MCFA system is characterized by robust, compact design and low consumption of the sample (72 µ L). The limits of detection for turbidimetric and nephelometric detection mode are 65 mg L(-1) and 9 mg L(-1), respectively. For turbidimetric measurements the range of linear response offered by the MCFA system is 72-900 mg L(-1), whereas in the case of nephelometric detection 18-500 mg L(-1) linear range is obtained. The throughput of the MCFA system is over 30 injection per hour. The analytical system was optimized with bovine serum albumin standards and successfully validated with real samples of human cerebrospinal fluid.

Trypan blue exclusion assay by flow cytometry

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Trypan blue exclusion assay by flow cytometry.

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Uncovering the proteome response of the master circadian clock to light using an AutoProteome system.

In mammals, the suprachiasmatic nucleus (SCN) is the central circadian pacemaker that governs rhythmic fluctuations in behavior and physiology in a 24-hr cycle and synchronizes them to the external environment by daily resetting in response to light. The bilateral SCN is comprised of a mere ~20,000 neurons serving as cellular oscillators, a fact that has, until now, hindered the systematic study of the SCN on a global proteome level. Here we developed a fully automated and integrated proteomics platform, termed AutoProteome system, for an in-depth analysis of the light-responsive proteome of the murine SCN. All requisite steps for a large-scale proteomic study, including preconcentration, buffer exchanging, reduction, alkylation, digestion and online two-dimensional liquid chromatography-tandem MS analysis, are performed automatically on a standard liquid chromatography-MS system. As low as 2 ng of model protein bovine serum albumin and up to 20 µg and 200 µg of SCN proteins can be readily processed and analyzed by this system. From the SCN tissue of a single mouse, we were able to confidently identify 2131 proteins, of which 387 were light-regulated based on a spectral counts quantification approach. Bioinformatics analysis of the light-inducible proteins reveals their diverse distribution in different canonical pathways and their heavy connection in 19 protein interaction networks. The AutoProteome system identified vasopressin-neurophysin 2-copeptin and casein kinase 1 delta, both of which had been previously implicated in clock timing processes, as light-inducible proteins in the SCN. Ras-specific guanine nucleotide-releasing factor 1, ubiquitin protein ligase E3A, and X-linked ubiquitin specific protease 9, none of which had previously been implicated in SCN clock timing processes, were also identified in this study as light-inducible proteins. The AutoProteome system opens a new avenue to systematically explore the proteome-wide events that occur in the SCN, either in response to light or other stimuli, or as a consequence of its intrinsic pacemaker capacity.

Development of bovine serum albumin certified reference material.

We present the development process for National Institute of Metrology (NIM) bovine serum albumin (BSA) certified reference material (CRM). Each CRM unit contains about 200 mg of purified BSA. The moisture, ignition residue, molecular weight, and high-performance liquid chromatography (HPLC) purity were analyzed and mass spectrometry based protein identification was carried out to ensure the material was BSA. Both amino acid based isotope dilution mass spectrometry (IDMS) and a purity deduction method were selected for value assignment. The certified value was the average of the IDMS and the purity deduction result. HPLC purity analysis was used to examine the homogeneity and stability of solid BSA CRM. Fifteen units were selected for between-bottle homogeneity examination and seven subsamples from the same bottle were selected for within-bottle homogeneity examination. Statistics showed the CRM passed both the between-bottle and the within-bottle homogeneity examination. The CRM stability under storage conditions (-20 °C) was tested for 18 months and no trend was observed. Uncertainties from the balance, amino acid purity, hydrolysis, method reproducibility, homogeneity, and stability were taken into account in uncertainty evaluation. The final certified value of NIM BSA CRM is (0.963±0.038) g/g.

Albumin-based or albumin-linked calibrators cause a positive bias in serum proteins assayed by the biuret method.

BACKGROUND: Assay of total serum protein by the biuret method calibrated with albumin standards according to the reference method provides results with a positive bias approximately 3%-5% exceeding the total error of 3.4% allowable for total protein in serum analysis made by analysers using two-part reagents and short-term procedures. METHODS: We used two types of two-part biuret reagents utilised in a short-term measurement in analysers with albumin or serum calibrators, in which protein was attested by the Kjeldahl method. RESULTS: Tests with potentially interfering substances proved that serum blanking used in a short-term biuret procedure is not capable of sufficiently eliminating effects of serum interferents. A short-term blanking is evidently capable of suppressing only an absorbance caused by serum-present coloured and turbid interferents, but its capacity to transform them (oxidise, hydrolyse, saponify, etc.) to some other not-interfering substances is very low compared with a long-term blanking. Lipids and bilirubin are responsible for significant positive bias of total protein in normal serum samples (approximately 3%) and even a greater positive offset in lipaemic and icteric sera (approximately 5%). We verified that interference tests based on a normal serum spiked with endogenous lipids and bilirubin give quite false and misleading results in the biuret reaction. A pure albumin, not depending on its bovine/human origin, gives absorbance responding only to its copper complexes with protein with a biuret regent, while its absorbance with a serum also includes the absorbance of interferents present in serum. The simplest way to improve current short-term biuret procedures is the use of a human serum calibrator with total protein attested by the Kjeldahl method. A serum calibrator, behaving analogously to serum samples, compensates for a positive bias in most normal sera. Reagents with a greater concentration of active biuret components (copper and alkali, reference method included) seem to be unnecessarily aggressive to proteins and are responsible for a lower accuracy when used in short-term measurements. CONCLUSIONS: Standard Reference Material 927c based on pure bovine albumin is still recommended and used as the primary standard for assays of total protein by colourimetric methods. The albumin calibrator is responsible for a positive bias of approximately 3%-5% in serum total protein assayed by the biuret reaction both in the reference and in current methods. Its substitution by a serum calibrator attested by the Kjeldahl method could solve this drawback. Clin Chem Lab Med 2009;47:91-101.

A systematic investigation into the recovery of radioactively labeled proteins from sodium dodecyl sulfate-polyacrylamide gels.

We report the results of a systematic investigation designed to optimize a method for quantifying radioactivity in proteins in sodium dodecyl sulfate-polyacrylamide gels. The method involves dissolving appropriately sized pieces of gel in hydrogen peroxide and heating to 70 degrees C overnight followed by liquid scintillation counting. H(2)O(2) had no effect on the count rates of [(14)C]bovine serum albumin (BSA) when counted in a conventional liquid scintillation system, and the count rates remained stable for several days. Temperatures below 70 degrees C resulted in incomplete extraction of radioactivity from gels containing [(14)C]BSA, but there was also a significant reduction in count rates in samples incubated at 80 degrees C. At 70 degrees C recovery was not affected by the amount of sample loaded onto the gel or by the staining procedure (Coomassie Brilliant Blue or SYPRO Ruby). Recoveries were in the range of 89-94%, and the coefficient of variation for five replicate samples was 5-10%. This method offers a reliable way of measuring the amount of radioactivity in proteins that have been separated by electrophoresis. It may be useful, for example, in quantitative metabolic labeling experiments when it is necessary to know precisely how much tracer has been incorporated into a particular protein.

The colorimetric detection and quantitation of total protein.

Protein quantification is an important step for handling protein samples for isolation and characterization; it is a prerequisite step before submitting proteins for chromatographic, electrophoretic, or immunochemical analysis and separation. Colorimetric methods are fast, simple, and not laborious. This unit describes a number of assays able to detect protein concentrations in the low microgram to milligram per milliliter ranges in a variety of formats.

Influence of gelatin and bovine serum lubricants on ultra-high molecular weight polyethylene wear debris generated in in vitro simulations.

Ultra-high molecular weight polyethylene (UHMWPE) wear debris induced osteolysis has a major role in the late aseptic loosening and ultimate failure of total hip replacements (THR). Clinically relevant in vitro simulations of wear are essential to predict the osteolytic potential of bearing surfaces in artificial hip joints. Newborn calf or bovine serum has been accepted as a boundary lubricant for such in vitro tests, but its biological stability has been questioned. This study compared the wear factors, number of wear particles and levels of microbial contamination produced in bovine serum and a gelatin-based lubricant. The wear factors produced by the two lubricants were not significantly different, however the wear debris morphology produced was substantially different. The bovine serum became contaminated with micro-organisms within 28 h, whereas the protein-based lubricant remained uncontaminated. The results showed that bovine serum was not a stable boundary lubricant. They also showed that although the wear factors for the two solutions were not significantly different, the protein-based lubricant was not a suitable alternative to bovine serum because the wear debris produced was not clinically relevant.

Acceptability, safety, and digestibility of spray-dried bovine serum added to diets of recovering malnourished children.

BACKGROUND: Specially collected, spray-dried bovine and porcine blood plasma have been incorporated previously in feeds of weanling farm animals, resulting in increased dietary intakes and greater rates of weight gain than observed in control animals. Before conducting similar trials in human populations, preliminary studies have been completed to assess the acceptability, safety, and digestibility of processed animal plasma in young children. METHODS: Masked study diets were provided sequentially to each of ten young, Peruvian children recovering from severe protein-energy malnutrition during three randomly ordered 7-day dietary periods. The control diet was prepared from rice, milk, vegetable oil, and sugar; the two study diets included spray-dried, bovine serum concentrate to replace either 25% or 50% of the milk protein of the control diet. Urine and feces were collected quantitatively during the last four days of each diet period to assess stool weight, apparent absorption of macronutrients, and retention of nitrogen. RESULTS: All children consumed the entire amounts offered of each of the diets. The mean number of daily bowel movements and mean apparent absorption and retention of nitrogen and mean apparent absorption of carbohydrate were similar for each diet. Fractional absorption of dietary lipid and of total energy increased significantly in relation to the amount of bovine serum concentrate in the diet, although this might be explained by the simultaneous replacement of milk fat with additional vegetable oil. CONCLUSIONS: Each of the diets was well accepted by the study children, and there was no evidence of any adverse effects of bovine serum concentrate.

The prevention of prosthetic infection using a cross-linked albumin coating in a rabbit model.

We evaluated the effects of a serum protein coating on prosthetic infection in 29 adult male rabbits divided into three groups: control, albumin-coated and uncoated. We used 34 grit-blasted, commercially pure titanium implants. Eleven were coated with cross-linked albumin. All the implants were exposed to a suspension of Staphylococcus epidermidis before implantation. Our findings showed that albumin-coated implants had a much lower infection rate (27%) than the uncoated implants (62%). This may be a useful method of reducing the infection of prostheses.

A procedure for total protein determination with special application to allergenic extract standardization.

A method for total protein determination of allergenic extracts has been developed and evaluated. Samples were hydrolyzed with 5 M NaOH followed by colorimetric determination with ninhydrin of the released amino acids using bovine serum albumin as the standard. The entire procedure was carried out in disposable plastic tubes. Substances (glycerol, phenol and mannitol) commonly present in allergenic extracts manufactured for human use did not affect the assay results. Analyses of four different pollen extracts by the method gave good agreement with amino acid analyses. Other methods of analysis (total N, protein N unit assay, Lowry) gave more variable results compared with amino acid analysis. Analysis of the total protein content of 53 different lots of allergenic extracts gave narrow ranges of values for each species. Standardized mite extracts analyzed for total protein by US FDA-licensed manufacturers using this assay showed a good correlation of biological activity with total protein.

Effect of zwitterionic buffers on measurement of small masses of protein with bicinchoninic acid.

Linear standard curves were obtained for 1 to 10 micrograms of bovine serum albumin measured in the presence of 50 mM Mes, Mops, Pipes, Caps, Bes, Hepes, Heppso, and Tes zwitterionic buffers using bicinchoninic acid for detection. The zwitterionic buffers Ada, Tricine, and Bicine that strongly bind Cu2+. Tris, and Bistris, each at 50 mM, inhibited color development to prevent quantitative measurement of protein mass.