2 replies to this topic

[Serendipitous]

Hi all,

I am going to attach my protein of interest with a tag for the purpose of ChIP. There are several tags such as FLAG, HA, MYC, GFP, Biotin, HIS, GST tags. From your experience and knowledge, which tags give good specificity with little background? What are the issues to consider? Thanks.

[rkay447]

Serendipitous, on Apr 23 2009, 05:11 PM, said:

Hi all,

I am going to attach my protein of interest with a tag for the purpose of ChIP. There are several tags such as FLAG, HA, MYC, GFP, Biotin, HIS, GST tags. From your experience and knowledge, which tags give good specificity with little background? What are the issues to consider? Thanks.

For immunoprecipitation, which is the first step in a ChIP, I've had great success with little or no background with myc and HA.  I also like these tags because they are small.  Tags can interfere with protein function, interactions, stability, localization, pretty much everything so I always try to work with small tags if possible.  I'm not a big fan of FLAG since I seem to get a significant amount of background.  Also, you can buy the myc already pre-conjugated agarose beads so it makes it much easier to do an equal IgG control (which you can also buy pre-conjugated to beads).  Just keep in mind that an IP will (most likely) work better if you have more than one myc or HA tag.  I had a robust IP with only one HA but was told that I had gotten lucky.  If you know what domain of the protein binds the chromatin (NT or CT) you would want to put the tag on the other side to help avoid disrupting binding abilities but it may not be an issue either way.

[LifeTein Peptide]

personally, i like gst. It is very easy to handle but it is big. GST at my hands gave me high yield all the time. I do not like His because of the bleaching problem. Cmyc is very good too. As the friend above said, it works great if you have 2 or 3 repeat cmyc tag.

James Chou
www.lifetein.com
Understanding life one protein at a time...