Hanging drop method

Figure 18.4 The hanging-drop method of protein crystallization, (a) About 10 pi of a 10 mg/ml protein solution in a buffer with added precipitant—such as ammonium sulfate, at a concentration below that at which it causes the protein to precipitate—is put on a thin glass plate that is sealed upside down on the top of a small container. In the container there is about 1 ml of concentrated precipitant solution. Equilibrium between the drop and the container is slowly reached through vapor diffusion, the precipitant concentration in the drop is increased by loss of water to the reservoir, and once the saturation point is reached the protein slowly comes out of solution. If other conditions such as pH and temperature are right, protein crystals will occur in the drop, (b) Crystals of recombinant enzyme RuBisCo from Anacystis nidulans formed by the hanging-drop method. (Courtesy of Janet Newman, Uppsala, who produced these crystals.)...

Figure 3 Protein crystallization diagrammatic representation of the hanging-drop method of vapor diffusion.

As described earlier, there are a number of different ways of crystallizing proteins. By far, the most common approach is the vapor-diffusion method, as mentioned earlier. Approximately, 70% of the crystal structures reported have been crystallized through variations of the vapor-diffusion method. The technique can be carried out in a number of ways the simplest two being the hanging drop method, and the sitting drop ... 

Mikol, V, Rodeau, J.-L. and Giege, R. (1990). Experimental determination of water equilibration rates in the hanging drop method of protein crystallization. Anal. Biochem. 186, 332-339. 

The embryonic stem cell test is an animal-free alternative test method for developmental toxicity. Mouse embryonic stem cells are cultured in a hanging drop method to form embryoid bodies. These embryoid bodies, when plated on tissue culture dishes, differentiate to form contracting myocardial cell foci within 10 days. Inhibition of cardiomyocyte differentiation by test compounds serves as the end point of the assay, as monitored by cormting contracting muscle foci under the microscope. 

Figure 3.2 Growing crystals by the hanging-drop method. The droplet hanging under the cover slip contains buffer, precipitant, protein, and, if all goes well, growing protein crystals.

One widely used technique is vapor diffusion, in which the proteinlprecipitant solution is allowed to equilibrate in a closed container with a larger aqueous reservoir whose precipitant concentration is optimal for producing crystals. An example of this technique is the hanging-drop method (Fig. 3.2). 

FIGURE 2.8. Some methods for growing crystals from solution, (a) Slow solvent evaporation, primarily used for small molecules, (b) Vapor diffusion, (c) The hanging drop method, primarily used for macromolecules, (d) A crystallization plate used for hanging drops there are slightly different crystallization conditions in each well, varied systematically with respect to, for example, pH and ionic strength. 

In this method, principally, the culture conditions for stem cells are changed from monolayer to suspension culture. There are three different types of methods, namely, suspension culture, hanging drop method, and forced aggregation method. 

The hanging drop method is shown in (a). It can be carried out in different ways but a typical procedure is the following ... 

Figure 6.10 Sitting Drop-Vapour Diffusion Identical principles to the hanging drop method (see Figure 6.9).

Interfacial tension measurements by the Hanging Drop Method. A bitumen droplet of known volume is formed at the tip of a hypodermic needle immersed in an aqueous solution. The shape of a drop of fiquid hanging from a syringe tip is determined from the balance of forces which include the surface tension of that liquid. The surface or interfacial tension at the liquid interface can be related to the drop shape through the Young-Laplace equation. (Hiemmenz, 1988)... 

Timmins NE, Nielsen LK. Generation of multicellular tumor spheroids by the hanging-drop method. Methods Mol Med 2007 140 141-51. 

Horsefield, R., 8c Neutze, R. (2006). Crystallization of lysozyme by the hanging drop method. http //www.csb.gu.se/rob/PDFs/Crystallisation Course 2006 Part-I.pdf. Retrieved 10 Mar 2011. 

Another rather reliable approach to measuring 7 is the hanging drop method (Adamson, 1990). After measuring the dimensions dg and d (the maximum diameter dg, and the diameter dg measured a distance dg above the bottom of the drop) from enlarged photographs of the drop, can be obtained as follows (Adamson, 1990) ... 

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