Protein Services


Protein Synthesis

We are utilizing the M15 strain of E.coli which is optimized for prokaryotic protein expression. This strain works by utilization of 2 different vectors. pQE is used for protein expression and pREP4 is for induction control. pQE is optimized for protein expression and purification by affinity chromatograph against HisTag by utilization of NiNTA columns. It codes for 6 histidine residues either on the N terminus or C terminus of the protein of interest according to your needs.

pREP4 is responsible for lacR expression and silencing the open reading frame of pQE. This strategy makes protein expression highly controllable and rescues E.coli cultivation from the effect of the expressed recombinant protein. E.coli is inoculated and cultivated until log phase at which time IPTG (a lactose analog) is administered.

IPTG binds the lac repressor and changes its conformation so that the lac repressor can no longer bind to the lac operon and the protein of interest is free to be expressed. This strategy enables maximum yield of expression of the protein of interest. This is especially critical for the expression of proteins that are toxic to E.coli. HisTagged protein purification can be performed under denatured or native conditions. This choice depends on the application in which the protein is going to be utilized. For proteins that are toxic to E.coli, it is possible that protein cannot be obtained using this strategy. In such cases, we perform in-vitro translation.




In vitro Translation

In-vitro translation is the expression of the target protein or proteins in a cell-free environment which has certain advantages and uses among molecular biology applications. In-vitro translation applications can be used in rapid identification of proteins, folding studies, production of proteins that are toxic to the host cells and that cannot be expressed via recombinant expression methods, incorporation of modified or unnatural aminoacids into the growing peptide chain and production of proteins that are prone to form inclusion bodies or proteolytic degradation.

In principle, in-vitro translation using cell-free extracts can be prepared from any kind of cells and give rise to the production of proteins from any mRNA molecule. In practice, cell-free extracts have only been developed from cells which have a high protein synthesis rate including rabbit reticulocytes or E.coli for eukaryotic and prokaryotic proteins, respectively.

E.coli is the most favored organism for large-scale protein expression and purification. However, some proteins cannot be expressed in E.coli as they are toxic to the organism. Furthermore, in order to express a protein in E.coli, the open reading frame coding the protein needs to be cloned into a suitable vector which is optimized for procaryotic protein expression. The plasmid then needs to be transformed into a suitable E.coli strain. These steps normally take about 3 days and the procedures are very labor intensive.

An alternative to this strategy is utilization of in-vitro translation. Bioneer’s ExiProgen is a fully automated system that can efficiently perform in-vitro translation of 16 different proteins simultaneously. Each protein is expressed with a HisTag and purified in a scale of up to 100 ug using a standard kit. The system is also suitable for up to 500 ug/protein expression and purification using its maxi kit. Alternatively, up to 9 disulphide bond-bearing proteins can also be expressed and purified by this system.

Western Blotting

For our western blot services, we use BioRad systems for vertical gel electrophoresis, semi-dry and wet transfer, imaging and documentation. Routinely used SDS-PAGE gels are produced in our lab. If desired, ready gels can also be used.

With Western blot, a host of applications are possible. For example, you can check the expression level of a protein of interest in cell lysates. Furthermore, the purity and identity of an expressed and purified protein can be checked.

Also, using Western blot, the presence and amount of expected antibodies in serum can be measured. The specific antibodies to be utilized in the Western blot procedure can be provided by Sentegen or by the customer as well. Our Western blot services also include a detailed report with analysis/discussion of the results.






Antibody Development

Coming soon: We will start offering custom primary and secondary antibodies for research. The antibodies, which can be used in western blotting, ELISA, MesoscaleTM, immunohistochemistry and immunofluorescence, will be developed rapidly in mice as monoclonal or polyclonal antibodies. When requested, the antibodies can be tested in western blot and provided with a guarantee to work.

The antigen or vector needed for the development of a primary antibody can be provided either by the customer or by Sentegen (with an additional fee and longer return time in the latter case). The product obtained after immunization can be provided either as serum or purified antibodies.

The time to shipment of the final product takes approximately 10-12 weeks for monoclonal antibodies, 6-8 weeks for polyclonal antibodies, and 5-6 weeks for serum. However, this waiting period may be longer for antigens with low immunogenicity and/or when the antigen/vector is developed or provided by Sentegen.


Q-dot antibody conjugations

Quantum Dot - antibody conjugates have wide in-vitro applications. Western blotting, protein array screening for variable purposes, immunohistochemistry and immunocytochemistry are common examples. If the antibody is provided or ordered by the customer, conjugation service is available. The Quantum Dots that are conjugated routinely in our facility are excited with UV/blue light (472 nm) and emit red light (625 nm).

For special purposes, Quantum Dots that emit in other wavelengths are also available for conjugation as specified by the customer. Conjugation takes 24 hours and is shipped to the customer the same day. Western blotting / Dot blot is available as a separate service upon request.

ELISA

We provide both direct and indirect ELISA services. We have experience with protocol optimization and statistical analysis of the results. We are capable of both performing ELISA using commercial kits and developing and optimizing custom assays. We provide a technical analysis result report together with the results of the service.

With this service, standard curve generation, antibody titration, cytokine/chemokine or the concentration measurement of a desired protein, Monte Carlo analysis for cut-off determination and implementation of variable statistical methods for sensitivity and specificity calculations is included.