Hello Artem, Yes, we also tried the Expi293 cells when they first came out. They also did not perform well. When you tested with your generic medium did you use their transfection reagent or good ol PEI?
I would be very interested to see if over the years if anything has changed as well. Best regards, Kelvin On Mar 15, 2020, at 10:38 PM, Artem Evdokimov <artem.evdoki...@gmail.com<mailto:artem.evdoki...@gmail.com>> wrote: Hi Kelvin We never tried with ExpiCHO, but the Expi-293 cells in a generic medium performed like a geriatric gerbil compared to the Old Faithful in the actual medium. Transfection was still close to 90% but yield suffered considerably. I am sure that with enough diligence the secret sauce can be reproduced. We focused more on vector design, instead. Notably, there were batches of Expifectamine that did not perform consistently. Also fresh Expi293 cells are not always the same, in my experience. But this was ~2 years ago so maybe Thermo made improvements in their production lines (I bet they did, this is a nice product and it ought to be a good money-maker for them) Artem - Cosmic Cats approve of this message On Sun, Mar 15, 2020 at 5:27 PM Lau Kelvin <kelvin....@epfl.ch<mailto:kelvin....@epfl.ch>> wrote: Dear Artem, By work poorly do you mean, not as well and are you referring to all Expi cells? We have adapted ExpiCHO cells to another medium and although we see drastic decrease from using the complete Expi system (x15 volumetric yield), it was still considerably higher than our previous cells in the same medium (x5 volumetric yields). Will soon try with ExpiSf9 and baculovirus to see if they also perform similarly. Best, Kelvin On Mar 11, 2020, at 5:49 PM, Artem Evdokimov <artem.evdoki...@gmail.com<mailto:artem.evdoki...@gmail.com>> wrote: Hello Bad news: Expi cells work poorly in non-Expi medium :( Good news: you can add SelenoMet and make partially seleniated protein, which is usually enough. Note that too much SeMet is pretty toxic, and that the toxicity is associated with BOTH enantiomers of the amino acid, so if you have the ability to use the L-SeMet pure as opposed to the more common L,D-mix then you can put in more. Either way, it works. Expect your yield to suffer. Question: did you try iodine incorporation? You can do it on the level of pure protein, or crystals... Artem - Cosmic Cats approve of this message On Tue, Mar 10, 2020 at 1:51 PM LAURA DEL AMO MAESTRO <lam...@ibmb.csic.es<mailto:lam...@ibmb.csic.es>> wrote: Dear ccp4 community, I am in my last 3 months of my PhD, working at the Structural Biology Unit at IBMB-CSIC in Barcelona. As my experimental phasing trials using various heavy metals failed so far, we ordered two months ago Expi 293 (Met-) expression medium for selenomethionine labeling (see the reference: https://www.thermofisher.com/order/catalog/product/A4096701#/A4096701. But we got now the notice that it will not be delivered until end of April. I had been working so hard on this project, and this medium is essential to finished with the experimental part of my thesis. Thus, I was wondering if someone in the community has a bottle (1L) of this expression medium without methionine that I could borrow, and which I would return immediately once our medium arrives. Also in case you have recommendations or other sources of a defined Se-Met medium for Expi293 cells, please let me know. With kind regards from Barcelona, and hope someone can help, Laura --- Laura del Amo Maestro - Proteolysis Lab SBU-Structural Biology Unit, IBMB CSIC Barcelona, Spain, Europe ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
