Skip to content

Epitope tags for ChIP

August 19, 2011

After a lot of searching I found that there is very little out there on what epitope tags are good for ChIP. From my personal experience I found that FLAG, MYC and STREP tags do not work well. In general it is good to avoid tags with lysines (explains why FLAG and MYC are not good) because lysine is the major target of formaldehyde crosslinking.  This also explains the need to do native ChIP for histone modifications.  A coworker had a nice table from a book titled Epigenomics (Fergunson-Smith, Greally and Martienssen Springer, 2009). They (Brinkman and Stunnenberg) give a list of epitopes they have used. They grade on a +/- scale. Myc gets a -. FLAG gets a +/-. HA, HSV, TY1 get a single +. 2x-TY1 gets a ++. ERalpha gets a ++. I went ahead and made a 3x-TY1 retroviral vector. Ultramers from IDT are great for PCRing long tags onto cDNAs.  You don’t even need to get them PAGE/HPLC purified even at lengths up to 150 bp. The TY1 antibody was purchased from Diagenode and is pretty cheap as far as antibodies go. The 3x-TY1 tag worked very well even when the protein of interest was expressed at pretty low levels. I also have word from another colleague that the bio-tag works well for ChIP but that requires introducing a second plasmid into your cells.

One more important thing to consider when using epitope tags for ChIP is expression level. Most vectors use a CMV promoter or some other ridiculously strong promoter and result in extreme over-expression on your target protein. One way around this is to make stable cell lines and use a clonal cell line that expresses at a closer to endogenous level. Another way is to use a vector with a weaker promoter. I find the PGK promoter is a nice option for lower levels of expression but it is probably still too high for many proteins.

Advertisements

From → Posts

14 Comments
  1. Subhradip permalink

    Do you have a sequence for the 3X TY1 tag? Is there any commercial vector that has this tag?

    Thanks

    Subhradip

  2. ethanomics permalink

    TY1
    EVHTNQDPLD

    3x TY1 tag DNA sequence
    GAG GTG CAC ACC AAC CAG GAC CCC CTG GAC GCC GAA GTC CAT ACA AAT CAG GAT CCT CTG GAT GCC GAA GTG CAC ACC AAT CAG GAT CCC CTG GAC GCT

    Get the antibody from Diagenode. It’s pretty cheap.
    http://www.diagenode.com/en/catalog/antibodies-3/protein-tag-52/product/ty1-monoclonal-antibody-183

    I don’t know of any commercial vectors but since I’m a nice guy, I can send you the one I constructed. It is a retroviral construct with a puromycin selectable marker driven by the PGK promoter, which is constitutive but not very strong in most cells. The TY1 fuses to the C-terminus of your protein. Email your shipping address to me at eford.dna -at- gmail com if you like and I’ll get it out to you. Otherwise just order an long oligo containing ‘restriction site-3X TY1-annealing sequence to your favorite gene’ and a reverse primer and PCR it up and clone it into your favorite vector. For the long oligo I recommend Ultramers from IDT. They’re cheap and you don’t need to have them PAGE purified even for 150mers.

  3. Thanks. It is pretty good.

  4. lesande permalink

    Hi Ethan,
    Could you please post the result from one of your ChIP-PCR results from a 3x-TY1 fused protein?
    Thanks
    Les

  5. ethanomics permalink

    Hi Les,
    I’d old data now. I’ll have to dig through my notebook and don’t have time now. Perhaps later this week. It does work well. I’m sure of that.

    Ethan

  6. Riyan permalink

    Hi Ethan,

    I tried many tags for ChIP, but it did not work. Could you share the 3xTY1 vector with me? I will give you my FedEx number. Thanks,

    Riyan

  7. Any further thoughts on this, especially on tags that work particularly well in plants? What about 2x- or 3x-FLAG? Any idea why TY1 and ERalpha are so rarely used?

  8. ethanomics permalink

    Any epitope with a lysine(s) are generally bad since this is the target for formaldehyde. I’ve seen plenty of papers where people use FLAG but in my hands it barely works where Ty1 and HA work really well. I have no idea about plants not being a plant person although, we’ll be finding out sometime in the future in our lab since we are at least 50% a plant lab in a plant center.

    I think Ty1 is rarely used just because it’s off people RADAR. FLAG was the second epiptope tag (HA was the first). FLAG works really well for non-x-linked protein IPs so it has a lot of history, a lot of vectors, every lab has it, a lot of protocols, a lot of references, etc. When something works people rarely want to change. But I think people have mistakenly assumed since it works well for non-x-linked procedures it will work well for x-linked ChIP.

    I wouldn’t use ERalpha since it is a transcription factor.

  9. Louise Docherty permalink

    Hi Ethan,

    Would you be willing to share the ChIP TY1 protocol for ChIP? I have tried a few tagged options for my protein of interest but have yet to find anything that works very well. I am going to try 3XTY1 as a bit of a last effort so it would be really helpful to me to be able to see under what conditions you managed to get it to work for you.

    Louise

  10. NIMA permalink

    Hi Ethan,
    I wonder if it is still possible for you to send me the 3xTY construct. I was going to make my constructs with 3xTag that I saw this.

    Cheers
    Nima

  11. Miro Nikolov permalink

    Hey Ethan,
    thanx for the info! Do you have experience with 2/3x TY1 at the N-terminus? I want to tag both termini of my protein and want to know if the antibody has any issues if it is at the N. Same goes for HA/V5.
    Thank you!

  12. Lee permalink

    Hi,
    Thanks for your introduction. I am wondering what’s your opinion on using 3xTY1 as epitope for both CHIP and Co-IP (protein and protein interaction, not with nucleic acids)? Do you think I can this epitope to do both IP experiments?

    Thank you.

  13. Lee permalink

    Hi,
    I am wondering can I use 3xTY-1 for co-IP (protein protein interaction) as well?

    Thank you

    • ethanomics permalink

      Should work fine. I have used it and it worked but it was a IP-western. I’m not sure about the purity vs FLAG-M2 which is great for IP-mass spec.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

%d bloggers like this: