Cell Culture Studies
The physical and pathological role a recombinant protein plays is often measured in cell culture. As mentioned previously, it is essential that the protein is functionally active and free of contaminants for the response to be informative. Numerous bioassay protocols are available. Contact the R&D Systems’ Technical Service Department if your protocol of interest is not posted.
|A: A fully extended DRG growth cone growing on a laminin-coated tissue culture plate in the presence of R&D Systems human beta-NGF (Catalog # 256-GF).
B: A collapsed DRG growth cone following treatment with R&D Systems recombinant human Semaphorin 6B/Fc chimera (Catalog # 2094-S6). The ED50 for this effect is typically 5 µg/mL.
|Wnt-7a inhibits Wnt-3a ability to induce alkaline phosphatase (AP) synthesis by MC3T3-E1 osteoblastic cells. Cells were treated with increasing concentrations of R&D Systems recombinant human Wnt-7a (Catalog # 3008-WN) in the presence of 10 ng/mL of R&D Systems recombinant mouse Wnt-3a (Catalog # 1324-WN) for three days.
Applications – In Vivo
Several animal models of human diseases are available to assist in discovering the specific roles a protein plays in that disease. Species cross-reactivity of many proteins allows elucidation of biological information from in vivo animal experiments even when using a non-species specific protein.
It is important to use carrier-free protein for injection so that the biological response invoked is a result of the protein of interest and not the carrier protein. Bulk quantities of protein are also available for in vivo animal research experiments upon request.
Applications – Binding Assays
Recombinant proteins are useful tools in understanding protein-protein interactions, such as receptor-ligand binding and extracellular matrix interactions. Therapeutic strategies often focus on these interactions. A common binding assay, known as a functional ELISA, involves the attachment of a recombinant receptor extracellular domain to a microplate, followed by the addition and detection of the ligand. Carrier-free proteins should be used when coating the microplate to avoid competition of the recombinant protein and carrier protein during the plate coating process.
Applications – Enzymatic Assays
Proteases hydrolyze the peptide bonds of a protein in the processes of protein degradation or protein activity modulation. Non-proteolytic enzymes such as phosphatases, sulfatases, and kinases are involved in cellular processes. Enzymatic assays detect the conversion of a substrate by an enzyme over a period of time. R&D Systems offers a wide selection of active recombinant proteases and enzymes, recombinant protease inhibitors and regulators, and fluorescent substrates characterized by activity and specificity.
When used in conjunction with a matched antibody pair (PDF), recombinant proteins are used to construct standard curves of known protein concentration from which sample values can be derived. The mass of protein is determined by A280 and/or a Coomassie assay using BSA as a standard. After reconstitution, it is advisable to aliquot the protein into appropriate vial sizes to avoid repeated freeze-thaw cycles, which may be detrimental to the protein. The biological activity of a protein does not affect its use in an ELISA. Some proteins, for which matched antibody pairs are available, have no demonstrable biological effect and are only available as ELISA standards.
Recombinant proteins can be used as positive controls in Western blots. It is important to remember that the apparent molecular weight of a recombinant protein is dependent on the glycosylation level and may be different from that of the native protein. The biological activity of a protein does not affect its use in this application. Some proteins used to derive antibodies have no demonstrable biological activity. These proteins are available as Western blot controls.
Recombinant proteins may also be used to pre-absorb antibodies for specificity testing of antibodies in many applications, e.g. immunohistochemistry.