Hanging drop

Fig. 11-16. Shapes of sessile and hanging drops and bubbles (a) hanging drop (b) sessile drop (c) hanging bubble (d) sessile bubble.

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.)...

Tropfehen, n. little drop, droplet globule small bead, -kultur, /. hanging-drop culture. 

It proved possible to crystallize the Fepr protein over a wide range of pH, 5.9-8.0, and PEG 8000 concentrations, using both sitting and hanging-drop techniques. The crystals used for the X-ray analysis were produced using the following procedure (29) ... 

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

In contrast, the coupling of electrochemical and spectroscopic techniques, e.g., electrodeposition of a metal followed by detection by atomic absorption spectrometry, has received limited attention. Wire filaments, graphite rods, pyrolytic graphite tubes, and hanging drop mercury electrodes have been tested [383-394] for electrochemical preconcentration of the analyte to be determined by atomic absorption spectroscopy. However, these ex situ preconcentration methods are often characterised by unavoidable irreproducibility, contaminations arising from handling of the support, and detection limits unsuitable for lead detection at sub-ppb levels. 

Batley [780] examined the techniques available for the in situ electrodeposition of lead and cadmium in seawater. These included anodic scanning voltammetry at a glass carbon thin film electrode and the hanging drop mercury electrode in the presence of oxygen, and in situ electrodeposition on mercury-coated graphite tubes. 

Bond et al. [791 ] studied strategies for trace metal determination in seawater by ASV using a computerised multi-time domain measurement method. A microcomputer-based system allowed the reliability of the determination of trace amounts of metals to be estimated. Peak height, width, and potential were measured as a function of time and concentration to construct the database. Measurements were made with a potentiostat polarographic analyser connected to the microcomputer and a hanging drop mercury electrode. The presence of surfactants, which presented a matrix problem, was detected via time domain dependent results and nonlinearity of the calibration. A decision to pretreat the samples could then be made. In the presence of surfactants, neither a direct calibration mode nor a linear standard addition method yielded precise data. Alternative ways to eliminate the interferences based either on theoretical considerations or destruction of the matrix needed to be considered. 

The hanging drop mercury electrode can usually be applied down to trace levels of 0.1 - 0.05 xg/l. 

Batley [28] examined the techniques available for the in situ electrodeposition of lead and cadmium in estuary water. These included anodic stripping voltammetry at a glass carbon thin film electrode and the hanging drop mercury electrode in the presence of oxygen and in situ electrodeposition on mercury coated graphite tubes. Batley [28] found that in situ deposition of lead and cadmium on a mercury coated tube was the more versatile technique. The mercury film, deposited in the laboratory, is stable on the dried tubes which are used later for field electrodeposition. The deposited metals were then determined by electrothermal atomic absorption spectrometry, Hasle and Abdullah [29] used differential pulse anodic stripping voltammetry in speciation studies on dissolved copper, lead, and cadmium in coastal sea water. 

A 35-year-old female complains of itching in the vulval area. Hanging-drop examination of the urine reveals trichomonads. What is the preferred treatment for trichomoniasis ... 

Spherical Diffusion. If, as it might happen, the electrode is spherical rather than planar (e.g. using a hanging drop mercury electrode), See Figure 19, Fick s second law should be integrated by corrective terms accounting for the sphericity, or the radius r, of the electrode ... 

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 ... 

Electrochemical impedance measurements of the physical adsorption of ssDNA and dsDNA yields useful information about the kinetics and mobihty of the adsorption process. Physical adsorption of DNA is a simple and inexpensive method of immobilization. The ability to detect differences between ssDNA and dsDNA by impedance could be applicable to DNA biosensor technology. EIS measurements were made of the electrical double layer of a hanging drop mercury electrode for both ssDNA and dsDNA [34]. The impedance profiles were modeled by the Debye equivalent circuit for the adsorption and desorption of both ssDNA and dsDNA. Desorption of denatured ssDNA demonstrated greater dielectric loss than desorption of dsDNA. The greater flexibility of the ssDNA compared to dsDNA was proposed to account for this difference. 

Although microbatch is the simplest method of crystallization, it is a relatively new technique and many experimenters still prefer to use vapour diffusion which has been around and has worked well for over 40 years. Hence, there has also been major development in automating and scaling down the quantities of sample using the popular vapour diffusion method (both sitting and hanging drops). An increasing variety of robots are available commercially. 

Dilution experiments involve revisiting the crystallization drops which may cause disruption to the trial. An alternative way of backing off without touching the trial drop can be achieved in hanging drops in the following way the coverslips holding the drops are incubated for some time over reservoir solutions that normally give many small crystals (Protocol 3.6). 

Figure 3.4 Transfer of a hanging drop from nucleation to optimal growth conditions. Modified from Chayen and Saridakis (2002), Acta Cryst. 0 58,921.

Dispense the hanging drops on the coverslips as usual by mixing the protein solution with the reservoir solution. Use the resen/oir without oil as your source of precipitant for all the drops. 

Figure 3.6 Application of an oil barrier to improve crystals in hanging drops, (a) A standard hanging drop trial (b) a trial containing an oil barrier. Modified from Chayen (1997b). J. Appl. Cryst. 30, 198-202, with permission from the lUCr.

The main technique employed to set up crystal screens is the vapour diffusion method, either in the hanging drop or sitting drop set up. This method is based on slowly concentrating the droplet solution against a reservoir solution of infinite volmne (ml scale) compared to the volume of the droplet ( xl scale, see Fig. 14.2). Other techniques based on diffusion or counter-diffusion in agarose gels (Biertmnpfel et al., 2002) can also be useful. The... 

---