Electrophoresis probe concentration

We use the BioRad Miniprotean II gel electrophoresis system for gel shift analysis. The labeling of the DNA fragment that is used as a probe is described for the Southwestern experiment. For binding reactions the probe is diluted with TE to a final concentration of (0.6-1.2) X 10 mol//u.l when 1 /il is added to the 20-fi] reaction, the final probe concentration will be 30-60 pM. This low concentration of probe is necessary to ensure that the protein is in excess over the DNA. This is an important consideration for quantitative gel mobility shift experiments, where approximate dissociation constants (Xd) are determined by using a fixed amount of probe and varying concentrations of protein. Under the condition of protein excess, the is a function of the protein concentration and independent of the DNA concentration and can be estimated from the amount of protein needed to achieve a 50% shift (Ekker et al, 1991). 

The diagnosis of P-thalassemia minor, with appropriate indices in the CBC, is dependent on the finding of a raised Hb A.2 concentration (>3.5%). Iron deplete individuals should become iron replete before a definitive diagnosis of P-thalassemia is made as the Hb A2 may be falsely low. HPLC is the preferred method for this quantification since densitometric scanning of the Hb A2 band on an alkaline electrophoresis gel is not recommended because of poor precision and accuracy. In 30% to 40% of all cases of P-thaiassemia minor, the Hb F will also be raised (>1.0%). The lifespan of the RBC may be reduced, and diabetics may show a lower Hb Ai compared with normal individuals with equivalent glycemic control. The P-thalassemia mutation may be identified either by Southern blot using mutation specific probes or by gap-PCR. 

Using this approach, vancomycin was first run in the electrophoresis buffer with a probe ligand, Fmoc-Gly-D-Ala-D-Ala (L), that showed high binding affinity for the target substrate, vancomycin. It was shown that the concentration of vancomycin affected the electrophoretic mobility of L (Fig. 

Melton et al. (1984) observed that RPA allows the detection of as little as 0.1 pg of mRNA, which is at least ten times better than SI analysis. Moreover, RPA are easier and more reliable. Low abundance mRNA are more readily detected than with Northern blotting and quantitation is more accurate. Protected RNA is fractionated by polyacrylamide gel electrophoresis which allows, due to its high resolution, the mapping of the 5 and 3 -ends of the transcripts or the exon/intron boundaries (Calzone et al., 1987 Kekule et al., 1990) and even small differences between probe and target, e.g., after mutations, by adjusting the RNase concentrations (Genovese et al., 1989 Takahashi et al., 1989). 

To control the extent of water contamination with substances of environmental concern it is necessary to develop rapid and sensitive analytical methods. Modem gas chromatography (GC) and Hquid chromatography, especially in combination with mass spectrometry (MS), capillary electrophoresis (CE), and capillary electrochromatography are the most suitable techniques for the analysis of both an individual component and a complex mixture. However, as a mle, the concentration of solutes to be analyzed in water samples is too low, in the range of nanograms to micrograms per liter, to inject a probe directly into a chromatographic analytical column and, therefore, pre-enrichment of water samples is required. 

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