I would like to tag a few proteins which are in the range of 15 kDa to 35 kDa and use them for cell-culture based experiments. GFP and its derivatives being bulky, are not an option for me, at least for the 15 kDa protein. Hence, I am looking for any available fluorescent tags with mol.wt. less than 15 kDa. If anyone is aware of them, kindly let me know. Thanks.
Tetracystein tags could be the answer to your problem...
https://www.thermofisher.com/fr/en/home/life-science/cell-analysis/cellular-imaging/high-content-screening/flash-and-reash.html
The tetracystein system looks interesting. Since this technique has been around for >10 years, I wonder why it is not used more? Why are people using, for example, the SNAP, HALO and CLIP system, even though those tags are much larger? Is the tetracystein labeling prone to a lot of unspecficity?
Since high-affinity interaction of arsenic for thiols is the basic concept of this technology, cell toxicity should be assesed.
Thank you all, for your inputs. I will get back after I read a little about Tetracystein system. Thanks.
Hi! I know it has been a while. I am also looking into a similar problem and I have found small mFAP tag:
Article De novo design of a fluorescence-activating β-barrel
Its a bit dim (35x dimmer than EGFP), but currently the authors have another version mFAP2b available, which is 4.4x brighter than the predecessor (so around 8x dimmer than EGFP), depending on abundance of your protein could suffice. Have you had any luck with other tags?
Maybe Y-FAST could be another option, which is only 14kDa. Meanwhile, Joanna Wardyn , where did you get the info for mFAP2b from? I cannot find it from internet. Thanks
Hello Fanning Zeng I will look up Y-FAST- thank you. So the authors of the paper I posted were describing the mFAP2 tag, when we requested the plasmid from them, they sent us both mFAP2 and mFAP2b. They said mFAP2b is an improved, new version. They are very kind I am sure they will share plasmids with you when you inquire. Good luck!
@Joanna Wardyn, would you kindly share your input on the mFAP2/mFAP2b? Did it work well? And @Wulligundam Praveen would you kindly share with us your input on the Tetracystein system?
Yes, there are fluorescent tags available with molecular weights less than 15 kDa, although the options are somewhat limited compared to larger fluorescent proteins like GFP. These smaller tags are usually not fluorescent proteins themselves, but rather small peptide tags that can be fluorescently labeled. Some examples include:
Each of these options has its own set of advantages and limitations, including differences in brightness, photostability, and the complexity of labeling procedures. The choice of tag will depend on the specific requirements of your experimental system, such as the nature of the protein of interest, the cellular environment, and the type of microscopy or detection method used.
l Perhaps this protocol list can give us more information to help solve the problem.
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