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 needs 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 provide about 2 ml protein buffer with declaration of all ingredients

Furthermore some information are essential for our experiments:

  1. date of the protein purification and storage/stability conditions
  2. image of SDS-PAGE and homogeneity verification (e. g. gel filtration)
  3. 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)

 

For academical use we provide for members of the Heinrich-Heine-University and external users the possibility to perform SAXS measurements. The CSS is equipped with a Xeuss 2 in-house SAXS device from Xenocs. The detector distance is variable from 250 mm to 2500 mm. The system is equipped with a Cu source with a wavelength of 1.5 Å. Samples can be measured in capillaries or small cells. For biological samples we offer the option to use a temperature-controlled flow cell where samples can be automatically injected via a temperature-controlled autosampler.

For a planned SAXS measurement, some aspects are of importance and some documents/chemical agents are needed (see also undefinedorderform):

  1. the low molar protein buffer should contain as less agents as possible (just as much as is required for protein stability; avoid phosphate buffer and more than 5 % glycerol)
  2. protein needs to be pure (at least 95 % on SDS-PAGE), and homogenous (only one oligomeric state)
  3. protein has to be stable over at least two days (at 4 °C, 12 °C or 20 °C)
  4. protein concentration at least: 5 - 10 mg/ml
  5. minimum protein volume per measurement: 70 µl
  6. please provide about 2 ml protein buffer with declaration of all ingredients. (Important: It has to be exactly the same buffer used for the protein purification/dialysis/concentration for proper intensity determination!)

Furthermore, some information are essential for our experiments:

  1. date of the protein purification and storage/stability conditions
  2. image of SDS-PAGE
  3. homogeneity verification (e. g. gel filtration)
  4. protein concentration, molecular weight, extinction coefficient and composition of the protein buffer
  5. if the protein requires certain ligands (like metal ions, cofactors etc.) for activity and/or stability, please state
  6. Protein sequence including purification tags and cleavage sites (text file)
  7. If available: structure or homology model

For further information or appointments please contact us.

 

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