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1.
Bradford assay is one of the most common methods for measuring protein concentrations. However, some pharmaceutical excipients, such as detergents, interfere with Bradford assay even at low concentrations. Protein precipitation can be used to overcome sample incompatibility with protein quantitation. But the rate of protein recovery caused by acetone precipitation is only about 70%. In this study, we found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay. So we developed a method for rapid protein quantitation in protein drugs even if they contained interfering substances. 相似文献
2.
《Analytical biochemistry》1987,167(1):86-96
We recently reported a highly sensitive assay for measuring protein in solution based on the capacity of glutaraldehyde-treated protein to bind silver. This assay has now been made more sensitive, with a lower limit of detection of 5 ng, and more reproducible by supplementing protein samples with sodium dodecyl sulfate (SDS) to reduce protein loss to glassware. Two procedures have been developed. In one, protein samples are supplemented with both SDS and Tween 20 to yield very steep protein dose-response curves, which allow for more precise protein determinations, and very stable color formation, permitting OD measurements to be made several hours after the assay has been completed. In the second procedure, protein samples are supplemented with SDS alone which results in a less steep dose-response curve and less stable color formation but makes the assay substantially more tolerant of interfering substances. Thus, proteins in most commonly used buffers can be assayed directly with the second procedure without the need for buffer exchange. The procedure of choice, therefore, depends on the type and concentration of interfering substance. Proteins in buffers totally incompatible with either assay procedure (e.g., those containing reducing agents) can be easily buffer exchanged by centrifugation through 0.2% SDS equilibrated, drained Bio-Gel P-2 beads. The clinical utility of this improved assay is demonstrated by the accurate quantitation of protein in 0.5 μl of samples of human cerebral spinal fluid. This assay should therefore prove especially useful when a limited amount of protein is available for quantitation. 相似文献
3.
A modification of the protein binding assay for cyclic guanosine-3',5'-monophosphate (cyclic GMP) is described that is more sensitive and less subject to interference by cyclic AMP than are previously published protein binding methods. The assay employs a purified binding protein from the fat body of the pupa of the common silkmoth, Bombyx mori. The dissociation constant of the binding protein for cyclic GMP is 4.3 nM. A protein kinase modulator protein isolated from the same species increases the binding affinity and capacity of the cyclic GMP binding protein and can be used to advantage in the assay for cyclic GMP. As little as 0.1 pmoles of cyclic GMP can be detected by this procedure. Changes in the level of cyclic GMP in the frog heart during the cardiac cycle were determined by means of the new assay. 相似文献
4.
A rapid flow cytometry assay for the relative quantification of protein encapsulation into bacterial microcompartments
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Bacterial microcompartments (MCPs) are protein‐based organelles that have been suggested as scaffolds for creating in vivo nanobioreactors. One of the key steps towards engineering MCPs is to understand and maximize the encapsulation of enzymes into the lumen of the MCP. However, there are currently no high‐throughput methods for investigating protein encapsulation. Here, we describe the development of a rapid in vivo assay for quantifying the relative amount of protein encapsulated within MCPs based on fluorescence. In this assay, we fuse a degradation peptide to a MCP‐targeted fluorescence reporter and use flow cytometry to measure overall fluorescence from the encapsulated, protected reporter protein. Using this assay, we characterized various MCP‐targeting signal sequence mutants for their ability to encapsulate proteins and identified mutants that encapsulate a greater amount of protein than the wild type signal sequence. This assay is a powerful tool for reporting protein encapsulation and is an important step towards encapsulating metabolic enzymes into MCPs for synthetic biochemical pathways. 相似文献
5.
Quantitative assay of the binding of small molecules to protein: comparison of dialysis and membrane filter assays 总被引:5,自引:0,他引:5
J E Lever 《Analytical biochemistry》1972,50(1):73-83
A rapid assay involving filtration through a nitrocellulose filter is described for the quantitative detection of a protein which specifically and reversibly binds a small molecule. This assay is quantitatively characterized by direct comparison with equilibrium dialysis. The filtration assay is highly sensitive and reproducible when applied to the binding of histidine by the J protein, a component of histidine transport. The effects of several variables on this method are examined. Also, an equilibrium dialysis procedure designed for optimal sensitivity and range in the assay of proteins by binding activity is described. 相似文献
6.
Flow cytometric analysis of immunoprecipitates: high-throughput analysis of protein phosphorylation and protein-protein interactions 总被引:3,自引:0,他引:3
BACKGROUND: Activation-induced protein phosphorylation can be studied by Western blotting, but this method is time consuming and depends on the use of radioactive probes for quantitation. We present a novel assay for the assessment of protein phosphorylation based on latex particles and flow cytometry. METHODS: This method employs monoclonal antibodies coupled to latex particles to immobilize protein kinase substrates. Their phosphorylation status is assessed by reactivity with phosphoepitope-specific antibodies. The amount of immobilized protein on the particles was analyzed by direct or indirect immunofluorescence with antibodies to nonphosphorylated epitopes. RESULTS: The assay allowed measurement of phosphorylation of multiple protein kinase substrates in stimulated T cells, including the zeta chain of the T-cell receptor, ZAP-70, CD3, CD5, SHP-1, and ERK-2, using 1-3 microg of total cell protein per sample. The assay provided high resolution of kinetics of phosphorylation and dephosphorylation. Interactions of protein kinase substrates with associated signaling molecules were demonstrated. CONCLUSIONS: The novel assay allows high-throughput quantitative measurement of protein modifications during signal transduction. 相似文献
7.
The bio-bar code assay is an assay for ultrasensitive detection of proteins. The main technical hurdle in bio-bar code assay development is achieving a dose-dependent, reproducible signal with low background. We report on a magnetic bead ELISA screening mechanism for characterizing antibody pairs that are effective for use in the bio-bar code assay. The normal isoform of prion protein was utilized as the target protein as dozens of antibodies have been developed against it. The development of an ultrasensitive assay for the detection of the various isoforms of PrP has the potential to enable significant advances in the diagnosis and understanding of transmissible spongiform encephalopathies, including transmission mechanisms, disease pathology, and potential therapeutics. With prion protein as the target, the magnetic bead ELISA identified pairs with high background and low signal in the bio-bar code assay. The magnetic bead ELISA was effective as a screening mechanism because it reduced assay time and cost and allowed for understanding of pair characteristics such as development times and signal-to-noise ratios. 相似文献
8.
Quantitative analysis of c-myc-tagged protein in crude cell extracts using fluorescence polarization
A fluorescence polarization (FP) assay was developed to determine the concentration of a c-myc-tagged recombinant protein in a crude cell extract. The basis of the assay was a competition between a c-myc-tagged protein and a fluorescein-labeled c-myc peptide for a c-myc antibody Fab. Fluorescein-labeled c-myc peptide produced a high-fluorescence polarization signal upon binding to the c-myc antibody, which can be inhibited in the presence of a c-myc-tagged protein. Quantitation of a c-myc-tagged protein was realized by measuring the decrease in fluorescence polarization. The observed IC(50) values in the competition FP assay were similar among all monomeric c-myc-tagged proteins tested, indicating that the interaction of the c-myc tag with the antibody was independent of the fusion protein sequence. The c-myc-tagged protein concentrations measured by FP were found to correlate well with values derived from a spike experiment and with values obtained by quantitative immunoblot. This assay was not perturbed by the presence of crude cell lysate, dithiothreitol or detergents, and worked with both native and denatured samples from several expression systems, including Escherichia coli, Pichia, insect cells, and mammalian cells. The assay under the current condition can detect as low as 0.05% expression level of c-myc-tagged protein with regards to total proteins, depending on the expression system. This assay is both quantitative and rapid (less than 15min) and is therefore suitable for the optimization of recombinant protein expression conditions as well as for the monitoring of protein purification procedures. 相似文献
9.
In situ measurement of HMG-CoA reductase activity in digitonin-permeabilized hepatocytes 总被引:1,自引:0,他引:1
M J Geelen J S Papiez K Girgis D M Gibson 《Biochemical and biophysical research communications》1991,180(2):525-530
An assay procedure for HMG-CoA reductase is described which allows rapid measurement of the activity of this enzyme in isolated rat hepatocytes. In a one step procedure digitonin permeabilizes the plasma membrane and at the same time HMG-CoA reductase activity is measured. Digitonin at a concentration of 64 micrograms per mg of cell protein was found to be optimal for exposing microsomal HMG-CoA reductase to the assay components. The enzyme assay is linear with time up til 5 min and with protein concentrations in the range of 0.06-0.6 mg of cell protein per assay. It is shown that cellular enzyme activity is affected by preincubation of intact hepatocytes with a variety of short-term modulators of hepatic cholesterogenesis. 相似文献
10.
Geelen MJ 《Analytical biochemistry》2003,322(2):264-268
An assay procedure for diacylglycerol acyltransferase that allows rapid measurement of the activity of this enzyme in isolated hepatocytes is described. The one-step procedure involves permeabilization of the plasma membrane with digitonin and simultaneous measurement of diacylglycerol acyltransferase activity. Digitonin at a concentration of 64 microg/mg of cellular protein was found to be optimal for exposing microsomal diacylglycerol acyltransferase to the components of the assay. The enzyme assay is linear with time up to 4 min and with protein concentrations in the range 0.25-2.4 mg of cellular protein/assay. It is shown that there is a good correlation of cellular enzyme activity as determined in digitonin-permeabilized hepatocytes with the rate of triacylglycerol synthesis in intact hepatocytes. 相似文献
11.
Assay of adenosine 3', 5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity 总被引:2,自引:0,他引:2
In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many 32P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of [γ-32P]ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of [γ-32P] phosphate from [γ-32P]ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: [ATP] = [ATP]0 e?[cAMP]kt. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the protein kinase stimulation assay based on transfer of [32P] phosphate from [γ-32P]ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use. 相似文献
12.
Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions 总被引:32,自引:0,他引:32
The bincinchoninic acid protein assay has been adapted for use with microtiter plates and a plate reader to reduce the time needed for analyses. The total volume of the assay was reduced to 0.21 ml with various ratios of sample to reagent volume being used. The assay is sensitive to a limit of less than 1 microgram of bovine serum albumin. The assay is insensitive to the presence of the detergents sodium dodecyl sulfate, Triton X-100, and deoxycholate. In addition, the method has been adapted for determining the protein concentrations of sucrose density gradient fractions, which eliminates the need for removing the sucrose prior to assay. 相似文献
13.
Xiao SH Farrelly E Anzola J Crawford D Jiao X Liu J Ayres M Li S Huang L Sharma R Kayser F Wesche H Young SW 《Analytical biochemistry》2007,367(2):179-189
Several drugs inhibiting protein kinases have been launched successfully, demonstrating the attractiveness of protein kinases as therapeutic targets. Functional genomics research within both academia and industry has led to the identification of many more kinases as potential drug targets. Although a number of well-known formats are used for measuring protein kinase activity, some less well-characterized protein kinases identified through functional genomics present particular challenges for existing assay formats when there is limited knowledge of the endogenous substrates or activation mechanisms for these novel kinase targets. This is especially the case when a very sensitive assay is required to differentiate often highly potent inhibitors developed by late-stage medicinal chemistry programs. ACK1 is a non-receptor tyrosine kinase that has been shown to be involved in tumorigenesis and metastasis. Here we describe the development of an extremely sensitive high-throughput assay for ACK1 capable of detecting 240 fmol per well of the kinase reaction product employing a BV-tag-based electrochemiluminescence assay. This assay is universally applicable to protein tyrosine kinases using a BV-tag-labeled monoclonal antibody against phosphotyrosine. Furthermore, this assay can be extended to the evaluation of Ser/Thr kinases in those cases where an antibody recognizing the phospho-product is available. 相似文献
14.
Summary An assay for the determination of the protein content of whole cells immobilized in cross-linked synthetic gels was developed. The assay is based on a three step procedure: a) methanol dehydration, b) protein extraction by 1.0 M alkali at 125°C c) colorimetric assay of the extracted protein according to Bradford's procedure (Bradford M. M. (1976), Anal. Biochem. 72:248–254). The procedure worked out was found adequate for the determination of the protein content of microbial cells immobilized in synthetic and native polymer-gel-systems. 相似文献
15.
Protein assay sensitive at nanogram levels 总被引:6,自引:0,他引:6
A simple, fast protein assay which utilizes the affinity of colloidal gold for proteins is described. This assay is sensitive at the 20-ng level when a visible light spectrophotometer is used to measure absorbance. Few chemicals interfere with the assay. Interfering reagents include those that are strongly alkaline, contain high levels of salt, or contain sodium dodecyl sulfate. The problem of alkaline interference can be overcome by acidifying the protein solution before performing the assay. Purified proteins have different capacities to interact with the colloidal gold but this variability is not greater than that seen with the Bradford protein assay. 相似文献
16.
The Coomassie brilliant blue assay for the determination of protein has been extended to rapidly and conveniently measure the protein concentration of cells growing in culture in a 96-well microtiter format. Modifications of the standard assay include sodium hydroxide to solubilize the cells and ovalbumin, instead of bovine serum albumin, as a protein standard. The procedure allows a large number of small samples to be assayed simultaneously. Two examples of its use, enzyme-specific activity and drug resistance, are shown. An assay for acetylcholinesterase activity in the same culture plate is demonstrated. G418, an inhibitor of cell protein synthesis, is frequently used to select for cells transfected with the neomycin resistance gene. The required concentration of G418 can be easily determined with this protein assay. 相似文献
17.
A solid-phase method for the quantitation of protein in the presence of sodium dodecyl sulfate and other interfering substances 总被引:4,自引:0,他引:4
We describe a simple assay for small amounts of protein that is insensitive to sodium dodecyl sulfate (SDS) or many common interfering substances including Tris and reducing sugars. For this reason, it is particularly useful in the analysis of protein content of samples prior to SDS electrophoresis. The assay consists of the following steps: (i) absorption of protein to nitrocellulose; (ii) fixation of the bound protein with methanol; (iii) staining of the bound protein with amido black; and (iv) elution and spectrophotometric measurement of the bound dye. The assay is sensitive to as little as 0.5 microgram of protein in 1 microliter of solution. Although SDS does not interfere appreciably with measurement, Nonidet-P40 does. 相似文献
18.
Staurosporine-based binding assay for testing the affinity of compounds to protein kinases 总被引:1,自引:0,他引:1
Staurosporine is a broad-spectrum inhibitor of both tyrosine and serine/threonine protein kinases. Excitation of staurosporine and its analogues at 296 nm results in major emission bands centered at 378 and 396 nm. The intensity of the emission bands is enhanced on binding to the adenosine triphosphate (ATP) site of many protein kinases. This property was used to develop a competitive displacement assay for evaluating the binding affinity of small molecules to protein kinases. The assay was validated in both cuvette and plate formats for several phosphorylated and non-phosphorylated protein kinases. The throughput of the assay is high enough to be used in drug discovery for screening as well as lead optimization. 相似文献
19.
Babu S. Antharavally Krishna A. Mallia Priya Rangaraj Paul Haney Peter A. Bell 《Analytical biochemistry》2009,385(2):342-112
We describe a dye-metal (polyhydroxybenzenesulfonephthalein-type dye and a transition metal) complex-based total protein determination method. The binding of the complex to protein causes a shift in the absorption maximum of the dye-metal complex from 450 to 660 nm. The dye-metal complex has a reddish brown color that changes to green on binding to protein. The color produced from this reaction is stable and increases in a proportional manner over a broad range of protein concentrations. The new Pierce 660 nm Protein Assay is very reproducible, rapid, and more linear compared with the Coomassie dye-based Bradford assay. The assay reagent is room temperature stable, and the assay is a simple and convenient mix-and-read format. The assay has a moderate protein-to-protein variation and is compatible with most detergents, reducing agents, and other commonly used reagents. This is an added advantage for researchers needing to determine protein concentrations in samples containing both detergents and reducing agents. 相似文献
20.
We have previously described a strategy for detecting protein protein interactions based on protein interaction assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA). Here we describe PCAs based on the enzyme TEM-1 beta-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10:1 to 250:1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quantitative analysis of a small molecule induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissociation constant and number of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of beta-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array analysis of protein protein or enzyme protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners. 相似文献