Can anybody help me with the points to be taken care of during cloning, expression and purification of a protein for the purpose of generation of antibodies against it?
The development of the antigen is one of the keys for the development of a good antibody. In this sense, it should take into account the technique for which the antibody is to be developed and in particular if the recognition of the target protein will be in native or denaturing conditions.
Based on these specifications, it will be possible to evaluate whether it would be sufficient to use a synthetic peptide as an antigen or a bacterial expression or even an expression in mammalian cells and the corresponding choice of tags for its purification.
In this sense and from my experience, a purity around 70% coud be enough for polyclonal antibodies develoment, while for a monoclonal it would be ideal to start with purities > 90%.
On the other hand, it is also important to take into account how much antigen you will need for the choice of production method. Thus, for example, the antigen quantity for a rabbit polyclonal immunization process could be around 3-3.5 mg, whereas for a mouse monoclonal it would enough with
First, write a detailed plan before you start. Choose the region of your protein before you start, decide if you will be making native or denatured protein, choose your purification tags, etc. Share your plan with your colleagues and ask for feedback. Do a literature search for similar proteins. Someone may have already made an antibody you can use!
Two, you need to sequence your expression plasmid. Trust me. Even if someone else made it and says they sequenced it. You don't want to discover in a year or two that your protein is mutated (or even the wrong protein).
Three, expect to run into issues (cloning trouble, protein is toxic to expression cells, protein is insoluble, etc.). Keep good notes.
Good luck!
If you have commercial antibodies (even a small amount) against your target protein, its good to confirm that your purified antigen is recognized by them. If it happens its likely that your protein could induce the formation of the desired antibodies. If not, its highly probable that your protein is not reproducing the natural epitopes.
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