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Protein crystallization and characterization

Protein crystallization and characterization

LP3 acts as a focal point for expertise and equipment for the entire process chain of production, purification, characterization and crystallization of proteins. At LP3, high-throughput, nanovolume characterization and crystallization of biological macromolecules is performed. LP3 enables users to carry out a wide variety of robot-assisted crystallization experiments. The success rate of initial crystallization trials is maximized by sampling many hundreds of conditions using minimal sample volumes. LP3 can also be used for designing and implementing optimization screens to produce crystals suitable for data collection.

Equipment

Crystallization

  • Freedom EVO 150 liquid handling workstation (Tecan)
    Used for generation of screens in deep-well blocks and dispensing into crystallization trays
  • Mosquito nanolitre pipetting robot (TTP Labtech)
    Allows the setting up of crystallization drops with volumes as low as 50 nl protein + 50 nl reservoir. Sets up 96 wells in around 2 minutes. Can set up 1–3 protein drops per reservoir, thus 96–288 experiments per plate.
  • Minstrel HT UV imaging system (Rigaku)
    Images the crystallization experiments at high resolution using visible and UV illumination. The latter allows the identification of even very small crystals hidden in precipitate, or discrimination of protein crystals from salt. Images are available to users over the Internet through the CrystalTrak software.

Biophysical characterization

  • Mx3005P qPCR system (Stratagene)
    Used for studying the thermal unfolding of proteins using the Thermofluor technique, otherwise known as differential scanning fluorimetry. Typically we use this to probe the effect of buffer conditions and additives on protein stability, which is useful in preparation for a crystallization experiment. The use of a reporter dye in these experiments means that they consume very little material.
  • Zetasizer APS dynamic light scattering system (Malvern)
    Used to study the aggregation state of proteins. The samples are stored in a 384-well plate at temperatures as low as 4°C and are analyzed in a quartz cuvette whose temperature can also be varied. Thus temperature- or time-dependent studies can be done on individual samples while the others are kept safe. We use this to investigate the effect of buffers, additives, time and temperature on protein aggregation. Samples that are not monodisperse are very unlikely to crystallize.
  • Leica microscope including 10.4 Megapixel digital camera

Booking, manuals and rules for use

The crystallization facility is available on Tuesdays - Fridays. A booking system is applied.

Time is bookable in 1-1.5 h blocks for the Mosquito, DSF for 3 hours and DLS is as needed.

Please contact maria.gourdon [at] biol.lu.se (Maria Gourdon) to book an instrument. We appreciate if you book a few days in advance.

Instrument availability can be checked using Google calendar, see link on the right hand side.

You will be required to fill in a sample sheet with the identity of your enzyme and other information.

If not stored by default in the Minstrel HT UV, your experiments can be set on marked shelves in the 12°C incubator and/or in the 4°C cold room.

Don't hesitate to ask for help if needed!

For manuals, please use the download links on the right hand side.

For the moment the most common screens are pre-pipetted in 96-well MRC or Rigaku plates and stored in the 12°C incubator (see below).

Available crystallization screens

From Molecular Dimensions

  • PACT Premier
  • JCSG+
  • ProPlex     
  • Structure Screen 1+2
  • Morpheus
  • MemStart & MemSys
  • Memgold
  • MemMeso
  • Stura Footprint / MacroSol
  • SG1
  • MIDAS

From Hampton Research

  • Additive Screen
  • Detergent Screen

We are constantly seeking to improve our service, so if there is a crystallization screen that you miss here, please let us know (well in advance) and we will try to provide it.

DSF Screens

 
Well Buffer Name
A2 Na acetate 100 mM (pH 5.0) & 150 mM NaCl
A3 Na MES 100 mM (pH 6.0) & 150 mM NaCl
A4 Ammonium acetate 100 mM (pH 6.0) & 150 mM NaCl
A5 Bis Tris HCl 100 mM (pH 6.5) & 150 mM NaCl
A6 Na cacodylate 100 mM (pH 6.7) & 150 mM NaCl
A7 Imidazole HCl 100 mM (pH 7.0) & 150 mM NaCl
A8 Na MOPS 100 mM (pH 7.2) & 150 mM NaCl
A9 PBS 12 mM (pH 7.4) & 150 mM NaCl
A10 BisTris propane HCl 100 mM (pH 7.5) & 150 mM NaCl
A11 Na HEPES 100 mM (pH 8.0) & 150 mM NaCl
B2 Na Bicine 100 mM (pH 8.3) & 150 mM NaCl
B3 Tris HCl 100 mM (pH 8.5) & 150 mM NaCl
B4 Na Glycine 100 mM (pH 9.5) & 150 mM NaCl
B5 Na HEPES 100 mM (pH 7.4)
B6 Na HEPES 100 mM (pH 7.4) & 50 mM NaCl
B7 Na HEPES 100 mM (pH 7.4) & 150 mM NaC
B8 Na HEPES 100 mM (pH 7.4) & 300 mM NaCl
B9 Na HEPES 100 mM (pH 7.4), 150 mM NaCl & 5% glycerol
B10 Na HEPES 100 mM (pH 7.4), 150 mM NaCl & 10% glycerol
B11 Na HEPES 100 mM (pH 7.4), 300 mM NaCl & 5% glycerol

The buffer screen is stored 4x concentrated and has been constructed similarly to the screen developed by Niesen et al., 2007. 3 volumes of sample (about 0.1 mg/ml) are mixed with 1 volume buffer screen, yielding the concentrations given in the table above.

Generally the protein is dissolved in 10 mM HEPES pH 7.5 with 1:1000 fold diluted SYPRO orange dye to a protein concentration of about 0.1 mg/ml. The wells of white PCR plates are filled with about 30 µl per well and the fluorescence in the wells is measured while increasing the temperature from 25 to 95 °C. The dye will bind to the protein as it unfolds the more hydrophobic surface is exposed the more the dye will fluoresce. It is possible to screen different buffer conditions, varying e.g. pH and NaCl concentration (see Niesen et al. 2007). It is also possible to screen for ligand binding to your protein. Stabilising additives like ions can also be found with this method, which works for most protein having a well-defined hydrophobic core. However the method often does not work for proteins with a large exposed hydrophobic surface in the native state, or for very small proteins.

F.H. Niesen et al., The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature Protocols 2: 2212-2221, 2007.

Prices (applies for LU users only)

External users should contact us for pricing.

 
  Prices
Crystallisation screens in 48-well or 96-well plate, 1-3 drops used 650 SEK
Temperature stability (Thermofluor/DSF), per plate / occasion 300 SEK
Dynamic light scattering, per plate / occasion 250 SEK
Expert assistance and analysis, per hour 280 SEK

A typical screen might consist of two general 96-condition screens (e.g. PACT Premier and JCSG+) conducted at two temperatures (typically 20°C and 4°C). A typical drop size consists of 100 nl of protein and 100 nl of precipitant solution. Each screen requires 15 µl of macromolecule. At 10 mg/ml this represents only 0.15 mg of sample per screen.

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Contact information

The LP3 labs are located on floor 1 in Biology building A, Sölvegatan 35, Lund.

The Biology Building

Lund Protein Production Platform
Biology Building A
Sölvegatan 35

Phone:
+46 46 2227785

E-mail:
lp3 [at] biol.lu.se


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Funding

LP3 acknowledges generous financial support from Lund University, Knut and Alice Wallenberg Foundation (SWEGENE programme), the Erik & Maja Lundqvist Foundation, the Carl Tesdorpf Foundation and the Swedish Research Council.