Sample Preparation


1.) Protein crystallization

For academical use for members of the Heinrich-Heine-University and external users we provide a widespread screening for crystallization conditions by means of the „sitting drop“ method, for soluble proteins as well as for membrane proteins. We are able to check about 2500 different conditions in an initial screening. Our service contains consultation, implementation, documentation and evaluation of the experiments over a 4 weeks period. Apart from screens with the apo-protein we can also set up co-crystallization trials with your protein plus substrate(s). For the screening we invoice incurred material expenses only (e. g. crystallization plates, sealing foil, screening kits, fine chemicals).

 

To ensure an optimal screening and a good reproducibility some aspects are of importance and some documents/chemical agents are needed (see also undefinedorder from):

  1. the low molar protein buffer should contain as less agents as possible (just as much as is required for protein stability; if possible avoid phosphate buffer)
  2. protein need to be pure (at least 90 % on SDS-PAGE), and homogenous (only one oligomeric state)
  3. protein has to be stable over at least three weeks (at 4 °C, 12 °C or 20 °C)
  4. protein concentration at least: 5 - 10 mg/ml
  5. minimum protein volume per screen (96 conditions): 15 µl
  6. please include in the delivery about 2 ml protein buffer with declaration of all ingredients

Furthermore some information are essential for our experiments:

  1. please declare date of the protein purification and storage/stability conditions
  2. please attach image of SDS-PAGE and of homogeneity verification (e. g. image gel filtration)
  3. please provide protein concentration, molecular weight and composition of the protein buffer
  4. if the protein requires certain ligands (like metal ions, cofactors etc.) for activity and/or stability, please state
  5. is there an activity assay? If so: please ensure that the protein is still active in the used buffer.
  6. In case of co-crystallization trials: please state the substrate(s) and suitable concentration(s). Include the substrate(s) in the delivery unless they are commercially available.

On request we offer further experiments, starting from optimization of the initial crystallization condition, searching for cryo-conditions for diffraction measurements and crystal characterization at the in house X-ray source up to data collection at a synchrotron and subsequent structure determination.

 

For further information or appointments please contact us.


2.) SAXS analysis (proteins)

The protein should be of high purity and homogeneity (only one oligomeric state) at a concentration of 2-5 mg/ml. Please provide a gel filtration profile and SDS-PAGE analysis. For the protein buffer a glycerol concentration above 5 % should be avoided. Known or possible ligands or substrates have to be delivered as powder since they need to be dissolved in an identical buffer. Please include information to suitable ligand/substrate concentrations.

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