Equipment

The Analytical Ultracentrifugation Lab is a research facility of the Biomedical Department at the Campus Drie Eiken of the University of Antwerp. The head of the faculity is Prof. Peter De Deyn. The contact person is Ing. Tony Aerts.

Techniques used

Our lab has a Beckman XLA - Analytical Ultracentrifuge ( AUC), equipped with scanning absorption optics (= from 190 to 800 nm). The temperature range is between 2°C and 37°C.

Sedimentation Equilibrium

By using this method, we can measure the molecular weight of the solute at one solute concentration. Using different concentrations of the solute we can have an idea of self-interaction or non-ideality. In this way we can calculate the association constant ( if there is one). Up to 7 samples can be analysed simultaneously.For the best results, we ask :

150 microliter solution of a concentration between 0.1 and 0.4 OD.Samples must be dialysed against the selected buffer, which should be free of absorbing components in the choosen wavelength range.We also need the extinction coefficient of a 1% solution of solute at the used wavelength.If known the partial specific volume is also important. 

Sedimentation Velocity

By using this method we can measure the sedimentation coefficient of the solute. This method will tell us something about the heterogeneity of the sample. For the best results we ask: 350 microliter solution with a concentration between 0.5 and 1 OD.

Publications

1) Coopman S.,De Block J., Aerts T., Peeters W.,Moens L. and Clauwaert J , ”Physical chemical studies on bovine eye lens proteins.II. Comparative physical study of the low-moleculair-weight alpha-crystallin from calf lens cortical and nuclear fiber cells”, in Exp.Eye Res. (1984) 38:463-476.,93,BP13.

2) Aerts T., Xia J.Z., Slegers H., De Block J. en Clauwaert J.,”Hydrodynamic characterization of the major intrinsic protein (MIP) extracted from the bovine lens fiber membranes in n-octyl-beta-D-glucopyranoside: evidence for a tetrameric structure “ , in J.Biol.Chem.(1990) 265,8675-8680.

3) Xia J.Z., Aerts T., Donceel K. and Clauwaert J.,” Light scattering by Bovine alpha crystallin proteins  in solution: Hydrodynamic structure and interparticle interaction”, in Biophysical Journal (1994) 66, 861-872.

4) Aerts T., Wang Q., Tatarkova S. and Clauwaert J.,”Physical chemical characterization of the different individuel cortical alpha-crystallin fractions from bovine lenses “ in Progr.Colloid Polym.Sci (1995) 99, 94-100.

5) Vanhoudt J., Aerts T., Abgar S. and Clauwaert J.,”Quaternaire structure and interaction parameters of bovine alpha-crystalline: influence of isolation conditions” in Progr.Colloid.Polym. Sci (1997) 107:88-93.

6) Vanhoudt J., Aerts T., Abgar S. and Clauwaert J.,” Quaternary structure of bovine alpha-crystalline : influence of temperature”, in International Journal of Biological Macromolecules  (1998) 22, 229-237.

7) Abgar S, Vanhoudt J., Aerts T. and Clauwaert J.,”Study of the chaperoning mechanism of bovine lens alpha-crystallin, a member of the alpha-small heat shock superfamily”, Biophys J., 2001 Apr;80(4):1986-95.

8) Abgar S, Yevlampieva N., Aerts T., Vanhoudt J. and Clauwaert J.,”Chaperone-like activity of Bovine Lens alpha-crystallin in the presence of dithiothreitol-destabilized proteins: characterization of the formed complexes”, BBRC,2000,vol 276,N:2,619-625.

9) Vanhoudt J., Abgar S., Aerts T. and Clauwaert J., “ Native Quaternary structure of Bovine Alpha-crystallin”, Biochemistry,2000,vol39,n:15,p4483-4492.

10) Abgar S., Backmann J., Aerts T., Vanhoudt J. and Clauwaert J. , “ The structural differences between bovine lens aA- and aB-crystallin.” In Eur.J. Biochemistry, 2000, 267,5916-5925.

11) Dewilde S, Kiger L, Burmester T, Hankeln T, Baudin-Creuza V, Aerts T, Marden MC, Caubergs R, Moens L., ” Biochemical characterization and ligand binding properties of neuroglobin, a novel member of the globin family.” In J Biol Chem. 2001 Oct 19;276(42):38949-55.

12) Filee P, Vreuls C, Herman R, Thamm I, Aerts T, De Deyn PP, Frere JM, Joris B., ” Dimerization and DNA binding properties of the Bacillus licheniformis 749/I BlaI repressor.” In J Biol Chem. 2003 May 9;278(19):16482-7.

13) Vreuls C, Filee P, Van Melckebeke H, Aerts T, De Deyn P, Llabres G, Matagne A, Simorre JP, Frere JM, Joris B.” Guanidinium chloride denaturation of the dimeric Bacillus licheniformis BlaI repressor highlights an independent domain unfolding pathway.” In Biochem J. 2004 Nov 15;384(Pt 1):179-90.

The system combines following components:

• Olympus BX61WI motorized fixed-stage microscope with a highly accurate Z-drive. 
• Andor DSD2 confocal device. Combining structured illumination and spinning disk technologies with a high sensitivity high dynamic range Andor sCMOS camera, the Revolution DSD2 produces image quality that typically exceeds confocal images captured with laser point scanning systems. The DSD2 captures images at high frame rates when imaging both fixed and live samples. With three confocal sectioning options, the DSD2 allows to trade optical sectioning with signal level and handles a broad magnification range and sample types from single cell to very thick specimens such as Drosophila embryos and Zebrafish. The design can image conventional transmitted light contrast techniques (e.g. phase contrast and DIC) to be combined with the confocal image.
• The following filter cubes are available: DAPI, GFP, RFP, Cy5
• Märzhäuser XY stage, carries the confocal microscope, with a ravel range of minimum 25 x 25 mm at a resolution of 0.01 μm the stage allows the scanning of the specimens in XY direction
  
• The combination of the Z-drive & the XY stage allow a stationary object table that is capable of handeling both small object glasses and large objects like our MEA 2100 amplifier.
• Imaris 3D and 4D Real-Time Interactive Data Visualization Software (3D reconstruction software)
  

A full motorised Scientifica upright microscope (model SliceScope) equipped with a scanhead (XY galvanometer) to achieve high-resolution and fast-scanning (positioning of a laser spot) across the field of view for scanning images or Opto-genetic stimulation.

The system is equipped with two power modulatable 25 mW lasers (488 nm &  561 nm).

Electrical recordings are made with a classic patch clamp setup or our MEA-1060 & MEA2100 in vitro recording systems.

The compact 64-channel eego amplifier is designed with mobility in mind. With 24-bit resolution, up to 5-hours of recording time and weighing less than 500 grams, eego sports offers complete freedom to collect high-density EEG data and a variety of physiological sensor data. 

Our MEA-1060 & MEA2100 systems can be used to record from neuronal or cardiac cultures, stem cells, or brain or cardiac slices. They are based on an idea from the 1970’s, when Thomas et al. found that electrical activity can be recorded extracellularly with microelectrode arrays (MEA). Over the decades, many peer-reviewed articles have proven that the MEA technology is indeed a very powerful tool in electrophysiology research and the technology around the MEA has improved significantly.

The MEA wells are transparent allowing optical stimulation both bottom up (LED) or top down (XY controlled laser) and Ca-imaging with simultaneous (synchronised) electrical recording.

Cells are cultured directly on the MEA or slices placed on it. The planar electrodes with a dimension of typically 10-30 µm are arranged in a square recording field where the spacing between these electrodes is in a range of 30-700 µm.

Addressing the specific research interests of the laboratory requires a multidisciplinary approach. Part of the research team's expertise and equipment is listed below.

Equipment for electrophysiology

Four high-resolution patch-clamp setups for analyzing  primary cell cultures or transiently/stably transfected cell lines, 2 of the setups are equipment with a pressurized fast-switching perfusion system for administering drugs/toxins.  

One cut-open vaseline gap oocyte setup able to perform state-of-the-art voltage-clamp fluorometry (VCF) experiments.  

Molecular biology and biochemistry techniques

Laboratory has a large collection of ion channel clones (mainly Kv channels) and the standard molecular biology equipment for cloning new channels or creating channel mutations.

Cell culture and expression systems

L2 cell culture facility equipped with two laminar flow hoods and 4 CO2 incubators. Common cell lines for heterologous expression of proteins (e.g. HEK293, CHO, Ltk-,…) are part of our routine cell culture repertoire. Cells can be transiently or stably transfected according to the experimental needs.

Facility to express channel proteins in Xenopus laevis oocytes (e.g. for performing VCF experiments).

The Portable-ME-System is a all-in-one solution for tethered in-vivo/ex-vivo recordings. It is the perfect solution for recordings from anesthetized animals or Langendorff heart preparations.

The signal is picked up by an electrode array, which is connected to a miniature preamplifier. Here, the signal is amplified for the first time. Then, the pre-amplfied data goes into the core element of the set-up: The Portable-ME-System. It includes  filter amplifier and data acquisition in one device and is available with 16 and 32 channels. From there, one USB-cable makes the connection to the computer, where the included data acquisition software Multi Channel Suite analyses the data.