Once a protocol for coating beads is developed and put in place, it will need to be scaled up in order to meet demand. Scaling up a process, however, requires careful attention to ensure that the details of the protocol are replicated for larger volumes.
As you will find in our protein purification handbook, choosing the right matrix for protein enrichment and purification processes, such as immunoprecipitation or pull-down assays, is an important step in optimizing the efficiency of a protocol. The decision will be based on a number of variables and ultimately depends largely on the nature of the target biomolecule. The goal is to choose a matrix that will not only maximize the final yield, but will also be practical and accessible.
It is well known that most purification processes of recombinant proteins are conducted through chromatographies of different types, mainly in column chromatography. Whether it’s by affinity chromatography, gel filtration or ion exchange chromatography, these already established methods are not exempt from limitations. Research in recent years has allowed us to develop a series of alternatives to chromatography that allow us to avoid many of these limitations.
This is the last of a series of posts on pros and cons of classical covalent links, which we have been publishing during the last few weeks. This one is about 3 well-known classical covalent links: streptavidin beads, biotin, protein A and protein G.
Most current protein purification methods use agarose beads carrying affinity functionalities such as IMAC, Glutathione, or antibodies. The choice of these functional groups depends on the protein of interest to be purified, and a large variety is available, including pre-functionalized beads that can be coupled to biomolecules (see SEPMAG® protein purification handbook chapter 4 and 5).
Merck Millipore has invited experts from around the globe to attend the symposium it has organised at the Hilton Hotel in Shanghai on May 15 and 16. The event will include contributors from China, as well as Germany, New Zealand, Russia, Spain and USA.
In purification of recombinant proteins, highly pure samples are rarely obtained with the initial stages of the process. Whether we perform a highly specific affinity chromatography (with histidine tags, for example) or purification with several stages of capture and intermediate purification, there are always contaminants in the final sample. As you will find in our protein purification handbook, these contaminants are molecules that are closely related to the protein to be purified since, if a high-resolution technique is not applied, they can be hardly differentiated from the protein to be purified.
This is the second of a series of posts on pros and cons of classical covalent links, which we will publish during the next few weeks. This one is about 3 well-known classical covalent links: tosyl, epoxy and chloromethyl groups. In the next posts we are going to review other examples, such biotin or streptavidin beads.
Affinity chromatography allows us to obtain good results and a high level of purity with a single purification step, since a structure that is exclusively found in the recombinant protein is used as a tag. However, this is not possible in all cases. There are proteins that don’t accept changes in their sequence, even if the changes are so minimal as the incorporation of a tag, since the proteins quickly lose their biological activity with any modifications in their structure.
This is the first of a series of posts on pros and cons of classical covalent links, which we will publish during the next few weeks. The very first one is about 3 well-known classical covalent links: carboxyl, hydroxyl and amino groups. In the next posts we are going to review other examples, such as tosyl or streptavidin beads.
