Off Belay forum


View latest messages in My Forums
This topic has been archived, and won't accept reply postings.

Topic - Q for bioscientists: CoIP with mCh & GFP tagged proteins?

Jimbo W on 07 Feb 2013
I have two human proteins A and B that I want to test the interaction between (by transient co-transfection in 293 cells):

Protein A I have as WT and multiple mutants that I want to test the interactions of with Protein B. I have these Protein A mutants tagged with HA, Flag and GFP tags.
GFP expresses, IPs and blots nicely. However, I've found that HA and Flag express well (as seen in lysates) but does not IP at all well, so the availability of these small tags appears to be low in the native state, whereas with the large GFP tag this isn't a problem

Protein B is tagged with mCherry - expresses, IPs and western blots nicely.

So the question is, can I combine mCherry tagged protein A and GFP tagged protein B? They are evolutionarily related, GFP from Aequorea victoria (Jellyfish) and mCherry a modification of RFP from Discosoma sp, but by eyeball, conservation is relatively low. So, the question is, do you think a GFP Ab will pull down mCherry or an RFP Ab pull down GFP? I'm hoping not!!

Comparison of GFP and RFP Sequences:

GFP MSKGEELFTGV VPILVELDGD VNGHKFSVSG EGEGDATYGK LTLKFICTTG -KLPVPWPT
RFP MRSSKNVIKEF MRFKVRMEGT VNGHEFEIEG EGEGRPYEGH NTVKLKVTKG GPLPFAWDI


GFP LVTTFSYGVQ CFSRYPDHMK QHDFFKSAMP EGYVQERTIF FKDDGNYKTR AEVKFEG
RFP LSPQFQYGSK VYVKHPADIP --DYKKLSFP EGFKWERVMN FEDGGVVTVT QDSSLQD


GFP DTLVNRIELK GIDFKEDGNI LGHK-LEYNY NSHNVYIMAD KQKNGIKVNF KIRHNI
RFP GCFIYKVKFI GVNFPSDGPV MQKKTMGWEA STERLYPRDG VLKGEIHKAL KLK---


GFP EDGSVQLADH YQQNTPIGDG PVLLPDNHYL STQSALSKDP NEKRDHMVLL EFVTAA
RFP -DGGHYLVEF --KSIYMAKK PVQLPGYYYV DSKLDITS-- -HNEDYTIVE QYERTE


GFP GITHGMDELYK
RFP GRHH----LFL

Thanks for any help!
... not showing 8 replies to this topic ...
Register as a New User or login to gain full access to the forums. Registration is quick and completely free.
If you are definitely logged on, press Ctrl+F5 keys to reload this page [read more]

Unregistered users can only read messages in the most recent topics.
ads.ukclimbing.com

This topic has been archived, and won't accept reply postings.

My Forums