PAGE Experiments

A variety of commercial apparatuses or gel boxes for vertical PAGE are suitable for native PAGE experiments. Temperature control is critical to the separation of RNA conformers (slow exchange), and thus the box must be designed for use with a circulating water bath. We have obtained good... 

We suggest that steps 1 through 6 be performed by the instructor prior to the beginning of the experiment. We perform this experiment in conjunction with Experiment 18 ( Western Blot to Identify an Antigen ). If this option is chosen, the protocol described above can be followed exactly. If the Western blot is not to be performed, the SDS-PAGE experiment described in Experiment 18 can be performed on a single set of samples (see step 16). 

Vertical gel electrophoresis apparatus, cold room or cold cabinet, Whatman paper sheets, gel dryer, phosphorim-ager screens (GE-Amersham), Phosphorimager (Typhoon or Storm - Molecular Dynamics), 10% (w/v) ammonium persulfate, TEMED and other accessories for normal PAGE experiment as mentioned above. 

A number of binding assays have been employed to study the binding of pheromones and odorants. A widely used assay is a native PAGE experiment where isolated binding protein is incubated with a different test compound that has been radiolabeled. After the incubation period, the sample is loaded onto a native PAGE,... 

On the basis of SDS/PAGE experiments, mitochondrial carnitine acyltransferase from mung bean hypocotyl is a monomer with a Mr of 45,000. Similar characteristics have been reported for CATs from mammalian mitochondria [1]. The apparent Mr of the native enzyme was estimated by gel filtration chromatography and was found to be 45,000. The values are consistent with a monomeric structure for the mitochondrial enzyme. The results are in accordance with those obtained for mammalian CATs but differ from those ontained for mammalian CPTs which are heteromeric enzymes exibiting very high molar masses and being composed of several subunits [1]. 

Experience has shown that is better to obtain basis sets in electronic form than paper form since even slight errors in transposition will affect the calculation results. Some basis sets are included with most computer programs that require them. There is also a form page on the Web that allows a user to choose a basis and specify a format consistent with the input of several popular computational chemistry programs at http //www.emsl.pnl.gov 2080/forms/basisform.htm. The basis set is then sent to the user in the form of an e-mail message. 

The functionality available in MedChem Explorer is broken down into a list of available computational experiments, including activity prediction, align/ pharmacophore, overlay molecules, conformer generation, property calculation, and database access. Within each experiment, the Web system walks the user through a series of questions that must be answered sequentially. The task is then submitted to a remote server, where it is performed. The user can view the progress of the work in their Web browser at any time. Once complete, the results of the calculation are stored on the server. The user can then run subsequent experiments starting with those results. The Web interface includes links to help pages at every step of the process. 

This book grew out of a collection of technical-support web pages. Those pages were also posted to the computational chemistry list server maintained by the Ohio Supercomputer Center. Many useful comments came from the subscribers of that list. In addition, thanks go to Dr. James F. Harrison at Michigan State University for providing advice born of experience. 

The feed-back design (Figure 6.3.3 on the next page) was a 2-level, 6-variables central composite plan that required 2 = 64 experiments for the full replica. A 1/4 replica consisting of 16 experiments was made with an additional centerpoint. This was repeated after every 3 to 4 experiments to check for the unchanged condition of the catalyst. The execution of the complete study required six weeks of around the clock work. In the next six weeks, mathematical analysis and model-building was done and some additional check experiments were made. 

On page 235-241 is the explicit solution used in Excel format to make studies, or mathematical experiments, of any desired and possible nature. The same organization is used here as in previous Excel applications. Column A is the name of the variable, the same as in the FORTRAN program. Column B is the corresponding notation and Column C is the calculation scheme. This holds until line 24. From line 27 the intermediate calculation steps are in coded form. This agrees with the notation used toward the end of the FORTRAN listing. An exception is at the A, B, and C constants for the final quadratic equation. The expression for B was too long that we had to cut it in two. Therefore, after the expression for A, another forD is included that is then included in B. 

On pages 3 and 4, the calculations for Chapter 5.3 Range Finding Experiments are calculated for the example. The results listed on page 4 in line 38 are listed again in Table 5.3.1 and shown on Figures 5.3.2 and 5.3.3. 

On page 4, rates are calculated for the four specified conditions. Variance is calculated in the experimental results and correlation coefficients are used to show that fraction of the variance in the experimental results accounted for by the model. This is over 99%. Finally the experimental error is calculated from the repeated experiments on page 5. 

A few topics have disappeared, and of course there are many gaps. One only has a finite number of pages and any text will naturally reflect the author s own experiences and preferences. Scattering theory and graph theory are both conspicuous by their absence, but there are several good texts on these subjects. 

---