Analytical ultracentrifugation service

Introduction

Analytical ultracentrifugation is a versatile biophysical method for in vitro analysis of proteins, nucleic acids and their complexes. The method was invented by Theodor (Thé) Svedberg in the 1920s. He combined the centrifugation with optical system to allow the detection of sedimenting particles. The unit of sedimentation velocity was named Svedberg [S] after the inventor.

The method can be applied to characterize the following features of proteins and protein complexes:

  • size distribution
  • shape
  • stoichiometry
  • purity
  • homogeneity
  • aggregation
  • homo- and heterotypic binding
  • binding affinities

Basis of the method

An analytical ultracentrifuge consists of an optical detection system integrated into an ultracentrifuge, allowing for the real-time detection of the evolution of the concentration distribution of particles subjected to centrifugation. Two major experimental methods are employed in analytical ultracentrifugation, which differ in the applied centrifugal force: sedimentation velocity (SV) and sedimentation equilibrium (SE).

An SV experiment is basically the observation of the free fall of particles in solution under the influence of a strong gravitational field: in the reference frame of the spinning solution column, the centrifugal force is equivalent to a gravitational force.

SE experiments are conducted in the same instrument used for SV experiments. These two methods are complementary and share many common practical considerations.

Obvious key differences are:

  • equilibrium conditions can be theoretically derived from equilibrium thermodynamics, without reference to the dynamics through which equilibrium is attained and therefore allow modeling the sedimentation behavior of solutions that may be kinetically intractable;
  • since there is no net transport at equilibrium, kinetic considerations and hydrodynamic friction are irrelevant for an equilibrium analysis;
  • in SE, lower rotor speeds are used such that the back-diffusion region imposed by the impermeable bottom reaches throughout the solution column;
  • the equilibrium experiment usually requires much longer times

(Source: Schuck, P., Zhao H., Brautigam C-A., Ghirlando R., (2016) Basic Principles of Analytical Ultracentrifugation. CRC Press)

Practical issues

Samples:

  • Protein concentration: 1-2 mg/ml

Volume:

  • 400 ul for sedimentation velocity (SV) experiments
  • 100 ul for sedimentation equilibrium (SE) experiments

Buffer:

  • phosphate buffered saline (PBS) if possible
  • ionic strenght: always use > 10 mM NaCl or other salt
  • try to avoid the use of glycerol and sucrose
  • low amounts of HEPES, Tris, DTT is allowed

Other requirements:

  • protein sequence including any tags/extra residues after cleavage (.txt)
  • protein mass corresponding to the sequence
  • PDB code, if available
  • composition of the buffer

 

Limitations:

Size range: 100 Da - 108 Da

KD: pM – mM

For further information :

https://www.beckman.com/centrifuges/analytical-ultracentrifuges/optima-auc/features

https://www.beckman.com/centrifuges/analytical-ultracentrifuges/optima-auc

https://youtu.be/fLKmzqlEdT4

Services

  • molecular weight determination
  • size distribution
  • characterization of shape
  • homogeneity analysis
  • purity
  • complex stoichiometry
  • characterization of aggregates

Rates

We can offer you service measurements at a fraction of the costs you would encounter in managing this method in your own lab.
In addition, you profit from our expertise and competence.
It is recommended to contact us about your scientific problem so that we can discuss the method you would need.

Academic service rates

Research groups at University of Debrecen and affiliated institutions on an overall cost-recovery basis.

  • 50,000 HUF / sample for a single problem analyis
  • for more complicated problems please contact us!

Commercial service rates

Analytical services are also provided to non-UD institutions, commercial and international clients. Commercial fees are competitive and compare favourably with costs for similar services from other laboratories.

  • 100,000 HUF / sample for a single problem analysis
  • for more complicated problems please contact us!

Procedure:

1. Please contact us by email (norbert.duro@gmail.com)

2. Discussion of the scientific problem and the most appropriate method for analysis (email, phone, skype)

3. Sample submission with the signed Sample Submission Form. Only the samples for which the Sample Submission Form was completed will be accepted for analysis!

4. The results will be sent to the Costumer electronically. The report has to be signed by the Costumer.

5. When the results are published, the service must be acknowledged:

"Analytical Ultracentrifugation for this work was carried out at the Laboratory of Protein Dynamics (LPD), Department of Biochemistry and Molecular Biology, University of Debrecen."

Sample submission

Samples may be delivered to the laboratory (University of Debrecen, Laboratory of Protein Dynamics,  Department of Biochemistry and Molecular Biology, Life Science Building 3.044., Egyetem tér 1., 4032 Debrecen) or sent by post/courier.

Samples should be submitted with a signed Sample Submission Form. Only the samples for which the Sample Submission Form was completed will be accepted for analysis!

Do not forget to email the amino acid sequence of the protein and the exact composition of the buffer!

Equipment

Optima AUC with Absorbance (ABS) and Interference (IF) optical detection system

Rotor: AN-50 (max. 50.000 rpm; ~200.000 x g)

https://www.beckman.com/centrifuges/analytical-ultracentrifuges/optima-auc

Contact

Prof. Monika Fuxreiter (group leader)
fmoni@med.unideb.hu

Norbert Duro (research fellow)
duro.norbert@med.unideb.hu