The Bioinorganic Research Lab at WITS University has 5 customized fume hoods for synthetic chemistry. Four of the hoods are large enough to accommodate a decent Schlenk line for inert atmosphere synthesis. Both high purity (dry) argon- and nitrogen-equipped Schlenk lines are present in the laboratory. The lab typically houses 8 MSc, PhD, and postdoc researchers. Because the lab is equipped for spectroscopy as well as molecular biology, the hoods are mostly adequate as not everyone carries out Schlenk-based synthesis all of the time. On occasion, sharing is necessary. Generally, we spend more time on biochemistry, biophysics, X-ray crystallography, molecular biology, and molecular simulations than synthesis. Once the target metal chelates have been made and are purified, they are ready for characterization and a host of other work.

The Bioinorganic Research Lab at WITS University is equipped with a basic PlasLabs nitrogen glovebox and independent vacuum desiccator. The glovebox houses a 4-decimal place balance to permit the weighing of hygroscopic solids directly into Schlenk apparatus or round-bottom flasks under nitrogen. The flasks are then stoppered and transferred to the Schlenk line for the addition of solvents and reagents using cannula transfer methods under nitrogen. The glovebox is not suited to work with highly oxygen-sensitive compounds.

The Bioinorganic Research Lab at WITS University is equipped with two new rotary evaporators (Buchi Rotavapor® R-100) to enable the heated removal of solvents under reduced pressure. These are essential instruments supporting compound synthesis in the group. We also have an old instrument (also a Buchi that has worked for > 25 years) in a fume hood for the removal of noxious solvents and reagents such as pyridine, thiols, and SOCl2.

There are numerous magnetic-stirrer hotplates with thermal heat-transfer blocks to enable the synthesis of compounds in refluxing solvents along with all the supporting glassware required for synthetic work and the work-up of chemical reactions (round-bottom flasks, beakers, Schlenk tubes, cannula needles, traps, Hirsch and Buchner funnels, and diaphragm pumps for vacuum filtration. Many metal chelates are not suitable for purification by column chromatography (unlike the ligands), so the group normally purifies metal complexes by recrystallization using several methods.

This is one of the most important instrumental methods for structure elucidation by the group. We use the School's X-ray diffraction equipment and contribute to its running and maintenance:

• Bruker Apex II (Mo Ka)

• Bruker D8 Venture (Photon II detector, Incoatec IuS Ag Ka, Mo Ka)

• Bruker D8 Venture Bio (Macromolecular X-ray diffractometer, Photon III detector, hybrid Diamond-Cu Ka IuS, hybrid Diamond-Mo Ka IuS, both by Incoatec, Germany). This instrument was installed in August 2021.

We do all of our own X-ray crystallography from small molecules to proteins. Students who wish to learn the methods are trained to solve and refine their structures to publication-quality models after completing the School's X-ray crystallography training module (MSc level). Facilities for growing, viewing, photographing, and mounting crystals for X-ray diffraction exist in the lab. We also have cryogenic transport apparatus (MiTeGen, USA) for synchrotron visits or studies at other labs in the RSA.

The Bioinorganic Research Lab at WITS University is equipped with a Snijders ULT freezer (-80 °C) for the storage of enzymes such as human topoisomerase I and II, bacterial DNA gyrase, etc. The system is equipped with a carbon dioxide backup cylinder.

The Bioinorganic Research Lab at WITS University is equipped with instruments for protein and nucleic acid electrophoresis. We favor equipment from BioRad and Invitrogen. Native and SDS PAGE gels for protein analysis are mostly run using commercial pre-cast acrylamide gradient gels. We hand-cast all agarose gels for nucleic acid electrophoresis (despite owning an Invitrogen e-gel system) as this offers more flexibility for experiment designs (e.g., running EB and non-EB format gels (EB, ethidium bromide). The gels, once developed, are stained then de-stained and imaged using a Syngene G:BOX Chemi XRQ gel doc system with a high resolution, cooled camera. We also use standard optical imaging for PAGE gels.

Importantly, we have a Merck-Millipore Direct-Q 3 water purification unit with UV sterilization to generate Type 1 water (ultrapure, nuclease-free) for molecular biology applications.

The Bioinorganic Research Lab at WITS University is equipped to perform PCR reactions (PCR = polymerase chain reaction). The Chair spent a lot of time researching PCR equipment suitable for our needs in medicinal bioinorganic chemistry and we chose to purchase a miniPCR Bio instrument (16 tube format) from miniPCR. Although not a real-time PCR instrument (which costs > ZAR 500k), the instrument is ideal for investigative research and we simply use agarose gels or our BMG FLUOstar plate reader to assay reaction products by absorption or fluorescence methods. We have used the system to carry out research-grade PCR experiments and can confirm that this is an outstanding instrument (in addition to being really cool in its design). The software is modern and excellent, giving students a real-time animation of strand separation, copying, and annealing steps throughout the PCR cycles.

The Bioinorganic Research Lab at WITS University is equipped with an Anton Paar rolling ball microviscometer. The instrument is used to measure the viscosity of DNA solutions (< 80 uL) in the presence and absence of added DNA-binding agents in order to elucidate the mechanism of interaction. From the dose-response profile of the DNA's viscosity with an added metallodrug and suitable controls (such as ethidium bromide for an intercalator, Hoechst 33258 for a minor-groove binder, and cisplatin for a base binder), it is possible to delineate the binding mechanism for a novel compound. When coupled with in silico simulations (Schrodinger, GLIDE, DFT) and electrophoresis data (EMSA and/or DNA cleavage assays), the combination of methods is powerful and gives excellent insights on the nature of the interaction(s) between a DNA target and a novel metallodrug candidate of interest.

The Bioinorganic Research Lab at WITS University is equipped with an AKTA Start instrument for protein purification by IMAC (immobilized metal ion affinity chromatography) and SEC (gel filtration chromatography). The instrument is a low-pressure system suited to purifying His-tagged proteins by IMAC, separating protein mixtures by SEC, and separating monomeric proteins from covalent oligomers. Supporting instruments include an Amicon ultrafiltration cell to concentrated fractions eluted from the column, a standard centrifuge (Hettich Rotofix 32A) for centrifugal concentration methods, a refrigerated high-speed centrifuge (Hettich Mikro 200R), and a range of dialysis cassettes, spin columns, and desalting columns (Sephadex G25).

Purified proteins are analyzed by mass spectrometry, CD spectroscopy, PAGE, electronic and fluorescence spectroscopy, and crystallized for single crystal X-ray diffraction studies.

The Bioinorganic Research Lab at WITS University has a Telstar LYO-ALFA -85 °C freeze dryer. This instrument is used to lyophilize proteins and DNA from aqueous buffers or deionized water for transport and storage. The unit may also be used after desalting a protein solution in order to concentrate it, though this is typically done using ultrafiltration methods through membranes with the correct MW cut-off to hold back the protein of interest. After ultrafiltration, lyophilization is quick and can be done in a microcentrifuge tube to obtain the dry protein for subsequent freezing/transport. [Note: some proteins and sensitive enzymes such as human topoisomerase I do not tolerate this treatment!]

The Bioinorganic Research Lab at WITS University is equipped with a Bruker Alpha FTIR spectrometer. The instrument is fitted with a thermostatic diamond ATR sampling attachment, has a frequency range of 375-4000 wavenumbers, resolution of 0.8 wavenumbers, a transmission attachment for solution state spectroscopy, and solution cells. A flow cell is available to record reactions in real time via an external reactor equipped with peristaltic pump solution transfer. The instrument is sensitive enough to record the IR spectra of DNA and proteins as well as metallodrugs bound to these targets.

Instrument. The Bioinorganic Research Lab at WITS University has South Africa's only magnetic circular dichroism spectrometer (also with fluorescence detection). The instrument is used to probe the structures of biomolecules such as DNA and proteins and to delineate their interactions with metallodrug candidates. The spectrometer is equipped with numerous accessories available for the JASCO J-1500 CD platform and was installed in November 2019. The instrument was funded by a successful NEP infrastructure investment grant application (67% NRF) and WITS University (33%).

Spectrometer Bench. The bench that the spectrometer is placed on was designed by Professor Munro to fit in the lab center and to perfectly accommodate the spectrometer and its accessories. The bench was custom built by the Chemistry & Physics Workshop out of stainless steel, aluminum, marine plywood, and sand-blasted shatterproof glass. Electrical conduit (steel) supplying power to the bench is located at the back and is wired into a 6 kVA UPS with an additional battery pack. The conduit was reclaimed from used material. The under-counter locker was custom-designed by Professor Munro and built out of marine plywood and stainless steel by Trade All Africa Engineering & Industrial (Pty) Ltd (Benoni, RSA). All of the under-counter units (which includes the UPS and chiller platform) are on 75 mm industrial castor wheels.

Comment. Why is any of this important? Because we feel our efforts to create the right bench to support the new spectrometer have yielded a functional and aesthetically pleasing result. Importantly, we saved a lot of money by building the infrastructure ourselves. The Chair would particularly like to thank Vincent (his stainless steel welding is very neat) and John from the Chemistry & Physics Workshop for their fine effort and skills.

The Bioinorganic Research Lab at WITS University is equipped with a Cary Eclipse fluorescence spectrometer with thermoregulation from 283 - 363 K. This instrument is especially important for measuring the steady state fluorescence (emission) spectra of luminescent and fluorescent metal complexes, organic ligands, as well as proteins and DNA. The instrument is routinely used to probe drug binding by proteins in the laboratory using the Stern-Volmer equation or other nonlinear methods. The instrument also has the ability to measure phosphorescent lifetimes (micro- to milliseconds) in solution and the solid state (298 K). Measurements at 77 K are not currently possible (we do not yet have a suitable cryostat).

The Bioinorganic Research Lab at WITS University has three dedicated UV-vis spectrometers which are used pretty much every day for carrying out molar absorptivity measurements, DNA and protein binding assays, chemical kinetics, and electronic structure determinations. All have thermoregulation from 283 - 363 K. The instruments are:

• SPECORD210 plus UV-vis spectrometer (200-1100 nm)

• Perkin-Elmer Lambda 365 UV-vis spectrometer (200-1100 nm)

• Agilent 8453 diode array UV-vis spectrometer (200-1100 nm)

We have also installed a BMG Labtech FLUOstar Omega multimode plate reader which has both UV-vis spectroscopy (200-1100 nm, diode array) coupled with filter-based fluorescence (F), time resolved fluorescence (TRF), and phosphorescence capabilities for assays in microplate format from 298 - 338 K in temperature. The importance of this instrument is that it is capable of many assays in 96-well plate format including ELISA and various TRF-based assays. Usefully, one can measure the binding of a metallodrug to a protein or DNA by both fluorescence and/or absorbance in triplicate (or more repetitions) just using a single plate and covering a nM to mM concentration range. The readout is fast (< 2 minutes per plate depending on the method). So by simply repeating the assay at 5 temperatures using the same plate and samples, full binding thermodynamics can be measured (using the van't Hoff equation) all in under 30 minutes. We are excited to finally have had the funding to purchase this instrument as it will dramatically cut down the time required to do ligand binding assays. [Students will, of course, need to be really careful with designing their experiments and with pipetting reagents into the wells during plate setup - this is key to getting high quality data from a plate reader.]