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What Are the Symptoms of Acrylamide Poisoning?. The initial symptoms of acrylamide poisoning on the skin are peeling of the skin at the point of contact, followed by tingling and numbness in the exposed area. If exposure by any means (touch, ingestion, inhalation) continues, muscular weakness, difficulty maintaining balance, and clumsiness in walking and in the use of the hands...
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WHICH GEL SHOULD YOU USE? SDS-PAGE, NATIVE PAGE OR ISOELECTRIC FOCUSING?. If you want to determine the molecular weight of your protein, use SDS-PAGE. Native PAGE is used to assay enzyme activity, or other biologi- cal activity, for example, during a purification procedure. Each kind of protein PAGE has issues to consider, and these issues are addressed in the next...
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Many non-ionic or zwitterionic detergents can be used for IEF or native PAGE to keep proteins soluble. CHAPS (3- [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) is most often used, as it is a very good solubilizer, and is nondena- turing. It should be used from 0.1% up to 4.0%. Another very effective solubilizer is SB 3-10 (decyldimethylammoniopropane- sulfonate), but it is denaturing (Rabilloud et al.,...
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How Do You Determine Molecular Weight on a Western Blot?. Use biotin-labeled molecular weight markers, and detect them with streptavidin-conjugated horseradish peroxidase or alkaline phosphatase. These markers will provide precise molecular weight values.. The pre-stained recombinant proteins of known, reproducible molecular weights discussed above can also determine the molecular weights of proteins on a blot.. Some researchers will cut off...
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After incubation with the sec- ondary reagent and washing, the blot is incubated with a substrate.. The enzyme catalyzes a reaction in which the substrate is con- verted to a colored precipitate directly on the membrane, essen- tially coloring the band on which the primary antibody has bound.. Those systems produc- ing extended light output have the advantage that several...
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Table 13.3 lists some common immunoglobulins and their reactivity. Why use Protein A and Protein G rather than a secondary antibody?. A species-specific secondary antibody will usually give stronger signal and better specificity than Protein A or G. The advantage of Protein A or G is versatility: the same secondary reagent can be used with a variety of primary antibodies....
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Background confined to the lanes is more likely to be related to non-specific antibody binding. If bands show up in the absence of primary anti- body, the problem can be assigned to the secondary antibody. With other problems the guiding principle is still the same: to try to glean as much information from the problem blot as possi- ble, to...
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Some nonradioactive detection systems allow for the quick inactivation of the enzyme that generates the signal, eliminating the stripping step prior to re-probing (Peterhaensel, Obermaier, and Rueger, 1998). The functional lifetime of fluorescent labels will vary with the chemical nature of the fluorescent tag and the methodology of the application. For example, signal duration of a fluorescent tag could be...
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The size of the nucleic acid being transferred, the physical characteristics of the membrane, and the composition of transfer buffer affect the transfer efficiency. There is no magic formula guaranteeing linear transfer of all nucleic acids at all times.. Linearity of transfer needs to be tested empirically with dilution series of nucleic acid molecular weight markers.. Membranes should be dry...
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What Are the Functions of the Components of a Typical Hybridization Buffer?. Hybridization buffers could be classified as one of two types:. Denaturing buffers are preferred if membrane, probe, or label are known to be less stable at elevated temperatures. Formamide concentration can be used to manipulate stringency, but needs to be >. formamide may be added to hybridization buffers....
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The fluors that are in proximity to the radioactivity fixed on the matrix will be activated by the radi- ation. These fluors give off light upon being activated, enhancing the signal coming from the radioactive sample. Fluorography requires film exposure at - 70°C for the same reason as required by intensifying screens.. The additional sensitivity provided by intensifying screens is...
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What Practices Should the Laboratory Use to Ensure That Storage Phosphor Screens Are Completely Erased before Exposure to a Sample?. This practice also mini- mizes any background signal on the screen due to prolonged storage in the presence of cosmic radiation or slight contamina- tion of the screen surface.. Storage phosphor imagers could generate misleading data if the screen was...
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K., Parker, B., and Kowalik, T. Lion, T., and Haas, O. M., Chakrabarti, S., and Mahmood, A. E., Surrey, S., Rappaport, E., and Fortina, P. M., Jerkovic, B., Arthanari, H., and Bolton, P. A., et al. Mineno, J., Ishino, Y., Ohminami, T., and Kato, I. Moichi, M., Yoshida, S., Morita, K., Kohara, Y., and Ueno, N. J., Rumney IV, S.,...
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effect on translation when they occur close to the initiator codon (Chen and Inouye, 1990). Secondary structures that occur near the start codon may block translation initiation (Gold et al., 1981. Buell et al., 1985), or serve as translation pause sites resulting in premature termi- nation and truncated protein. These can be found using DNA or RNA analysis software. Structures...
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Products should be analyzed by agarose gel electrophoresis to determine if DNA of the predicted size was inserted in the vector. As an alternative, PCR can be done using as template a small scraping from a colony on the plate.. Amplification of the plasmid DNA contained in the cells using the same primers used in cloning, or primers that anneal...
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Boyd, D., and Beckwith, J. R., Semon, D., Escanez, S., and Kawashima, E. Bula, C., and Wilcox, K. Negative effect of sequential serine codons on expression of foreign genes in Escherichia coli. Burton, N., Cavallini, B., Kanno, M., Moncollin, V., and Egly, J. Expres- sion in Escherichia coli: Purification and properties of the yeast general tran- scription factor TFIID. Suppression...
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In this new age of genomics, many of the genes we obtain are. Before embarking on an expression project you will need to locate a cDNA copy of the gene of interest. It is also possible in theory to express genomic DNA containing introns, provided that the expression host will recognize the proper splice junctions. In practice, however, this is...
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The strength of EF-1a and CMV was derived from a comparison to the RSV LTR involving stable expression of various monoclonal antibodies and tPA (Trill, 1998 unpublished).. As with the promoters, there are a number of sources of polyadenylation regions. CMV Human 4 Boshart et al. RSV Rous sarcoma 2 Gorman et al. Beta-actin Human beta-actin ND Ng et al....
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For example, some researchers have found that the use of large soluble tags such as GST can enhance the solubility of certain proteins, which favors the production of active protein (Davies, Jowett, and Jones, 1993. Weiss et al., 1995. Ciccaglione et al., 1998). We have also observed that the additional C-terminal immunoglobulin Fc fusions sometimes result in the enhanced production...
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IMPLEMENTING THE BACULOVIRUS EXPERIMENT What’s the Best Approach to Scale-Up?. There are several types of media that can be used, including serum-free preparations and formulations specifically made for T.. Cells grown in the presence of serum may require a weaning period before being adapted for growth in serum-free media. For the expression of cytoplasmic proteins, all types of media will...