Hello,
I am currently reading this article by Lin et al, 2020 (https://science.sciencemag.org/content/367/6473/100) about this TTC5 enzyme and its new-found role in tubulin mRNA regulation.
I am trying to understand the first figure (especially Fig. 1B), which explain and exemplify the experimental strategy that led them to study the TTC5 enzyme. There are a few things I do not understand about Fig. 1B (SDS-PAGE and autoradiography).
1) I was expecting to see a 10kDa band (corresponding to the nascent S35-labeled 95-mer chain alone). I am however surprised to see instead a 25kDa band, which according to the article, corresponds to non–cross-linked tRNA associated nascent chain. Why are tRNAs still associated with the nascent chain? I was expecting SDS-PAGE conditions to dissociate tRNAs from the nascent chains, no?
2) All other things being equal, the band that corresponds to TTC5 crosslinked to the nascent chain is roughly 100kDa. TTC5 is 50kDa. S35-labeled 95-mer chain is 10Kda. There is a delta of 40Kda, that I don't understand at all. Can any of you understand this?
I am probably missing on something... These experiments are definitely not my cup of tea, but I'd really love to understand this paper. Would somebody be willing to help me?
Thank you very much in advance,
Quentin
Quentin, Please check supplementary information. I quickly overlooked this paper as per your questions. They specifically mentioned that there are cytosolic chaperones that interact with the transmembrane domain of the translation product. Also, these are cross-linked products that they are talking about - cross-linking of your protein might end up so many different protein bands because your protein will interact with other proteins, and here they used UV. They are not pure protein which they crosslinked in first fig. They did mass spec to identify which bands correspond to the protein of interest.
Quentin, UV - activated crosslinking is irreversible. For example, in CLIP technique the one way to rid of the crosslinked protein is to digest it using proteinase K.
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