I am curious about the tagged western blotting, where, proteins are probed on a nitrocellulose/PVDF membrane with HIS-tag/GST-tag primary antibody initially, and later with a corresponding secondary antibody. I have never worked with tagged proteins/ antibodies before and would like to gain some preliminary knowledge about this particular method of western blotting. Thank you!
To start with tag protein, first study what tag you want to use, meaning His, GST , Flag. etc.
In a simple way you want to know. Using the above vector you first need to clone your protein of interest( Any protein of interest,), check whether it is cloned in the right direction, once you confirm it( Vector+Your protein of interest) transfer the cloned vector+ Your protein of interest in the cells and check for protein like normal western blot. If you have your protein cloned in the right direction or right allingnment (or if no cloning you will observe a band of his tag(which will be very small only contains His taq or Flag ,or GST if no cloning occured) and a bigger band which contains tag+your protein of interest, if you succesfully cloned your protein).
This tagged protein you can detect with western, again you can immnunoprecipitate your protein of interest and purify.
Good luck
Tagging proteins is useful for many reasons:
1. Really good antibodies are available for most tags, therefore if your protein is difficult to detect, tagging is one way of making this easier.
2. Because good antibodies exist for tags, this also make immunooprecipitation experiments easier as the protein is easier to pull out of the complex mixture of endogenous proteins.
3. Certain tags (like GST) can be cleaved off after purification so you can essentially purify and obtain large amounts of any protein that has this tag.
4. Depending on the size of the tag, the protein size and be altered (usually bigger) allowing you to differentiate between endogenous and overexpressed versions of the protein (good if you want to do some mutagenesis studies)
Be aware that tagged proteins do not necessarily behave as the endogenous, especially when overexpressed, so they aren't particularly good for understanding the function of a protein within a cell at normal physiological levels. So I guess to tag or not to tag depends on what you want to acheive at the end.
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