Sidec combines cryo-electron microscopy and unique resolution enhancing software to perform Protein Tomography™.
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Jack Elands
Phone: +44 7947 870 482
jack.elands@sidec.com
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Protein Tomography™ makes it possible, for the first time, to study individual proteins and complexes in solution, in cells and in tissue. This makes it possible to compare tomograms of proteins and their complexes between samples obtained from solution or tissue.
Protein Tomography™ has been applied to drug discovery and development for understanding target mechanism of action, in developing biologically relevant assays and model systems, developing therapeutic antibodies and for the development of patent application claims, etc.
Protein Tomography™ is based on electron tomography data, and is basically the molecular biology equivalent to medical imaging.
Protein Tomography™ provides information that shows the biomolecules of interest and their environment. Tomograms can be obtained from both in vitro and in situ samples. Soluble proteins can be in a buffer solution or in a cellular context. Membrane proteins can be visualized in the membrane setting, together with their extra cellular and intracellular domains.
Protein Tomography™ is used to study conformations and interactions of proteins. Hence, the method is well suited for studying e.g.:
Protein Tomography™ has been used to study small proteins and protein domains. However, peptides are too small for Protein Tomography™ studies.
The location of glycosylated sites can be approached by binding lectins to the target protein and determining where the lectins bind. The lectin is are large enough to be distinguished from the target protein.
Sample preparation techniques are designed to minimize artifacts. In vitro samples are flash-frozen in a millisecond to maintain the natural shape of the proteins. This prevents crystallization of water in the sample, thus minimizing distortion of the shape of the proteins.
In situ samples are fixed, cryo-sectioned and immunolabeled using established techniques.
When using antibodies to mark in situ samples, the tissue is chemically fixed before addition of primary antibody, thus locking the proteins in their present conformations.
If the specific influence of an antibody upon the protein conformation is to be studied the samples may be fixed after antibody addition. It is important to note, that studies in solution do not require gold-labeling, and hence no antibodies are bound to the protein of interest.
An antibody that is specific for the target molecule. Affinity purified polyclonal antibodies are preferred; however, monoclonal antibody preparations have been used successfully. The important point is the specificity of the primary antibody. Sidec supplies the gold-labeled secondary antibodies.
For in vitro samples (purified protein), no preliminary experiments are usually needed. For in situ samples (cells/tissue), a sample validation/optimization study is undertaken at Sidec to ensure that the sample is optimally prepared for Protein Tomography™ experiments. During this step, the antibody binding is validated, and the expression level and localization of the protein in the cells/tissue is determined.
Sidec recommends a sample purity of 75% or more. Impurities make it more difficult and time-consuming to identify the protein of interest, which can extend the project timeline.
A Protein Tomography™ analysis requires only small quantities of sample material. A total of 25 μl is needed for processing. Sidec recommends a sample concentration of 1 mg/ml. For more information regarding in situ samples, please contact us.
Sidec has standard procedures which are simple and effective. Sidec scientists confer with each client on sample preparation issues to ensure that the best samples are selected and that they are optimally prepared. The shipping procedure is routine.
For further information, please contact us using this form.