Gels with Cutting

In the previous chapter, starfish-shaped gel robot was prototyped. In this case, the gel was unconsciously cut. Let us reconsider the effect of cutting by comparing deformation response to square-shaped gel and cross-shaped gel. 

The shape of the gel was changed from beam to square plate. Then problem arose that the deformation response became uncertain. This problem was solved by shape design. First, the experimental setup, problem statement and solution are described. Then we discuss the meaning of the result. 

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Desalting and depigmentation of urine sample was carried out by gel-filtration on Sephadex G25 with cut mass 10 kDa. 

The work that follows pertains primarily to actin networks. Many proteins within a cell are known to associate with actin. Among these are molecules which can initiate or terminate polymerization, intercalate with and cut chains, crosslink or bundle filaments, or induce network contraction (i.e., myosin) (A,11,12). The central concern of this paper is an exploration of the way that such molecular species interact to form complex networks. Ultimately we wish to elucidate the biophysical linkages between molecular properties and cellular function (like locomotion and shape differentiation) in which cytoskeletal structures are essential attributes. Here, however, we examine the iri vitro formation of cytoplasmic gels, with an emphasis on delineating quantitative assays for network constituents. Specific attention is given to gel volume assays, determinations of gelation times, and elasticity measurements. 

Cover the dry gel with a protective plastic foil, cut it into 5 mm strips and store airtight at -20 °C (strips are stable for several months). 

Cut grooves (s) in a layer of agarose gel with a scalpel blade or forceps to accommodate the embryo. 

Preparation of strips IPG gels are cut off to obtain the strips of desired length (e.g., 3-5 mm in width) for 2-DE. The width of strips is important to obtain separation with high resolution. For instance, the strips of 18-24 cm provide high resolution, but those with 4-11 cm are suitable only for rapid screening. There are also commercially available strips in different widths. 

Run gels at constant voltage, 25-30 mA per slab gel, until the bromophenol blue marker dye has readied the bottom of the gel (about 1 hour). When completed, turn off and unplug the electrophoresis apparatus and remove the gel. Carefully cut the gel between lanes 5 and 6 so that you have two identical halves. Cut a notch in the corner of each gel half to indicate proper orientation. The half containing lanes 1-5 will be stained with Coomassie Blue to detect the location of all proteins. The other half (lanes 6-10) will be blotted onto a nitrocellulose membrane to analyze for glycoproteins. 

Fig. 3. Second dimension with several IEF gels Several IEF gels are cut, 0.5 cm above and 0.5 cm below the isoelectric point of the protein of interest They are placed side by side at the top of the second dimension slab gel Thus, only one SDS gel is needed to collect several spots of interest...

After the transfer period, turn off the power supply and disconnect the leads. Remove the cathode and uppermost three layers of filter paper. Cut the comer of the transfer membrane above the cut corner of the gel. Alternatively, mark the transfer membrane by tracing the gel with a soft lead pencil. 

Run 50 pi of a double-stranded PCR amplification in a low melting point agarose gel. After electrophoresis, cut the desired double-stranded product from the gel with as little excess agarose as possible. The gel band should contain approximately 200 ng of DNA (a good strong band by ethidium bromide fluorescence). 

Stain and visualize gels as above. If needed, take samples of individual alleles by cutting small pieces of the gel with a clean scalpel or razor blade. Avoid cross-contamination. Place cut pieces in 250 pi of LTE buffer. 

The membrane proteins isolated using the colloidal silica method described above are especially difficult to solubilize, and difficult to separate by 2D gel electrophoresis. Consequently the membranes were solubilized in Laemmli buffer with sonication in a 60 °C water bath, and separated by 1D S DS gel electrophoresis. This ID gel was cut into 1 mm bands, each of which was subjected to trypsin digestion. The peptides were recovered, separated and analyzed by LC/ESl MS/ MS. This strategy is illustrated in Eigure 13.5. More than 3200 peptides were... 

Cut away a 5-mm broad edge all around the gel with the razor blade. This portion will have suffered from heating owng to the high voltage. 

Remove the gel assembly from the vertical gel apparatus, separate the glass plates, and cut off the stacking gel with a sharp scalpel blade. Also cut off the separating gel beyond the dye front, and mark the gel uniquely for future orientation by cutting the comer (s) appropriately. 

Cut away any excess nitrocellulose from around the gel with a sharp scalpel blade the nitrocellulose should be marked in an identical manner to the gel. 

Remove the glass capillaries from the bulk ofthe gel by cutting around them with the use of a scalpel. Hold the individual glass capillaries under running tap water (hot) to remove the gel cylinders from within. 

Proteins separated by SDS-PAGE (Chapter 16) can directly be coated to plastic by diffusion from the gel following electrophoresis. The gel is incubated for 15 min in water at 37°C. The gel is cut into 1 mm slices which are incubated (in separate wells) with a 20-25-fold excess of carbonate buffer for 24 h at room temperature. 

As is mentioned above, the syneresis rate increases with increasing temperature and decreasing pH. A look at Eq. (17.17) shows that the total outflow of whey is proportional to the surface area of the gel and inversely proportional to the distance over which the whey has to flow. Cutting the gel into small pieces thus very much enhances the syneresis rate (in fact, syneresis is almost imperceptible before the gel is cut). The equation contains the endogenous syneresis pressure psyn. However, for a system that can show structural rearrangement leading to syneresis, any externally... 

Grabar and Williams (G5) have developed the following procedure for purification of the commercial product. Agar is dissolved by boiling in distilled water when a 6 % sol is attained, it is poured into wide trays after setting, the gel is cut into small blocks these are extracted for 2 days in distilled water, which is renewed 5 times. The gel is now perfectly white and is kept in stoppered sterile containers. In order to eliminate organic substances, Wieme (W4) extracts the gel with 50 % ethanol in distilled water. An exacting method of purification (Bl) uses electrodialysis. 

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