Here is a protocol about use of S2s and SeMet labeling from the ccp4bb (the crystallographers' friendly bulletin board for knowledge exchange). I suggest you read the thread starting from the bottom to follow the logic. There will be useful information there.

[log in to unmask]" type="cite"> Hi Xianchi and Engin,

My lab uses S2 insect cells extensively and we have

SeMet labeled proteins with them. We grow our cells at 23 oC not

at 28 oC. I performed protein expression studies of various

proteins and obtained more protein at 23 oC vs 28 oC. Just and

FYI tip for S2 cells and the Insect Select system.

Engin is spot on (shout out to the U of C crowd). We use the

methionine deficient 921 media from Expression Systems and

supplement it with SeMet. Here is the link.

http://www.expressionsystems.com/?mvcTask=productDetail&id=63

I will provide a short protocol that we use with the assumption that

you have a stable S2 cell line (a modified protocol could be used for

transient expression). We use shake flasks for are large cultures

in a refrigerated shaker/incubator. We have only tested this protocol with

secreted proteins into the culture media using pMT-BiP A transfer vector.

1) Subculture the cells to 100 mL of volume in S2 complete media

supplemented with 10% HI-FBS.

2) After the 100 mL culture grows for 3-5 days to a density of 5-10 x 106 cells/mL,

spin the cells, aspirate off the FBS medium, and resuspend the cells

in 500 mL of prewarm 921 delta media supplemented with 50 mg/L of sterile L-SeMet,

0.1% Pluronic F-68 in appropriate shake vessel, and let the S2 cell grow for 2 days.

3) On day 2, induce the S2 cells with CuSO4 (500-700 uM) and grow for an additional

5-7 days. Add fresh sterile 50 mg/L of L-SeMet to the culture every 24 hours.

We harvest and purify the SeMet protein using the protocol as the non-SeMet labeled

protein. We achieve 85-95% of SeMet labeling with this protocol.

We use 50 mg/L of L-SeMet, but you can also use 100 mg/L of D/L-SeMet.

Let me know if anyone has further questions.

Cheers,

Scott

*****************************************

Scott T. R. Walsh, Ph.D.
Assistant Professor
University of Maryland College Park
Institute for Bioscience and Biotechnology Research

Dept of Cell Biology and Molecular Genetics
Rm 3127E CARB-2
9600 Gudelsky Drive
Rockville, MD 20850
email: [log in to unmask].edu
phone: (240) 314-6478
fax: (240) 314-6255

On Dec 22, 2016, at 7:52 PM, Engin Özkan <[log in to unmask]> wrote:

Nothing specific about S2 cells to contribute... However, S2s usually grow fine in other insect media, such as those intended for lepidopteran cell lines like the Sf9 and High Five. Protocols established for those cell lines, for example, check out those using the medium ESF-921, would probably work for you. Please let us know what works out.

Engin

On 12/21/16 6:15 PM, Xianchi Dong wrote:

Dear All,

Does anybody have experience with SetMet protein in S2 cells? What kind of reagent should I use and is anything else I should pay attention to?
Thanks in advance.

Best,
Xianchi