Epitope tagging

Nelson, R. W. Jarvik, J. W. Taillon, B. E. Tubbs, K. A. BIA/MS of epitope-tagged peptides directly from E. coli lysate Multiplex detection and protein identificatin at low-femtomole to subfemtomole levels. Anal. Chem. 1999, 71, 2858-2865. 

Arden JR, Segredo V, Wang Z et al. Phosphorylation and agonist-specific intracellular trafficking of an epitope-tagged K-opioid receptor expressed in HEK 293 cells. J Neurochem 1995 65 1636-1645. 

In this chapter, we describe our methods for purification of eIF2B holo-complexes as well as the catalytic subcomplex and the catalytic eIF2Bfi subunit (encoded by GCD6 in yeast) alone. We typically epitope tag only one eIF2B subunit, over-express the desired subunit combination, and use a single affinity chromatography step to purify the desired complex. In early work, a... 

Figure 3-1 A method to GFP epitope tag a gene of interest. (A) Diagram representing the C and N terminal cassettes. The upstream and downstream primers representing 55 nucleotides upstream and downstream (but not including the stop codon) are depicted by I H and respectively, and represent the amplification sequence com-...

Knop, M., Siegers, K., Pereira, G., Zachariae, W., Winsor, B., Nasmyth, K., and Schiebel, E. (1999). Epitope tagging of yeast genes using a PCR-based strategy More tags and improved practical routines. Yeast 15, 963—972. 

The use of pairs of matched spectral variants of GFP, like cyan and yellow or GFP and mRed, to differentially tag proteins and look for interactions, is now in routine use. The approach can be readily applied to homodimerization of molecules that differ only by their living color or epitope tag. For example, homomultimer-ization of a viral coat protein can be observed by imaging a mixture of cyan and yellow tagged homomeric molecules [46], whereas oc-synuclein stacking can be detected by utilizing a-synuclein transcripts that encoded different epitope tags for detection by immunostaining [47],... 

The advantage of such co-purification protocols is that the fully processed protein serving as the bait can allow interactions in a native environment and cellular location to allow isolation of multicomponent complexes. One limitation with this approach is the necessity for an antibody with specific immunoreactivity and immunoprecipitative capability for the bait protein. This drawback can be addressed by expression of the protein with an epitope tag. Excellent antibodies to a variety of epitope tags are available and can be utilized for immunoaffinity purification. Tags such as 6-histidine and GST allow purification using affinity characteristics to nickel and GSH beads, respectively. 

Chen YIG, Maika SD, Stevens SW (2006) Epitope tagging of proteins at the native chromosomal loci of genes in mice and in cultured vertebrate cells. J Mol Biol 361 412 419 Kolodziej PA, Young RA (1991) Epitope tagging and protein surveillance. Methods Enzymol 194 508 519... 

We have also used mutagenic primers to insert histidine tags and epitope tags into cDNAs of interest (Neish et al, 2003). Inserting several codons into a cDNA is a little more difficult than simply substituting one amino acid for another, but is quite feasible using this technique. Optimization of the reaction is absolutely critical when performing more difficult manipulations such as this. 

H2A.Bbd was discovered as an H2A-like protein encoded by human ESTs [77]. H2A.Bbd is only 42% identical to conventional H2A and is smaller due to a shortened C-terminus that lacks the ubiquitination site that is present in most other H2As (Fig. 6). H2A.Bbd appears to be associated with nucleosomes as indicated by the co-purification of an epitope-tagged H2A.Bbd with nucleosomal fragments in a sucrose gradient run at high ionic strength [77]. 

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