2 replies to this topic

[Benita]

Dear all,

I used to transfect my cells in 15cm diameter culture plates with 80% confluence using Fugene 6. My protein is secreted into the media so I have to change the media (from serum containing - [better growth an healthy cells] to serum free media [no fcs background proteins in my purification])  after transfection. So I'd like to ask what, in your opinion, is the best timepoint. Some people telling me "the next day" (so 12-18h) or others told me 4-6h after transfection.

I'm not sure. If I change it earlier maybe there is more protein in the medium because they live longer. But maybe the FCS is important for the first 12h of protein building? I don't know. What do you mean?

Greetings from Germany.

Franzi!

Edited by Benita, 08 June 2010 - 04:18 AM.

[labrat612]

Benita, on Jun 8 2010, 05:50 AM, said:

Dear all,

I used to transfect my cells in 15cm diameter culture plates with 80% confluence using Fugene 6. My protein is secreted into the media so I have to change the media (from serum containing - [better growth an healthy cells] to serum free media [no fcs background proteins in my purification])  after transfection. So I'd like to ask what, in your opinion, is the best timepoint. Some people telling me "the next day" (so 12-18h) or others told me 4-6h after transfection.

I'm not sure. If I change it earlier maybe there is more protein in the medium because they live longer. But maybe the FCS is important for the first 12h of protein building? I don't know. What do you mean?

Greetings from Germany.

Franzi!

Hi Benita,

I'm going to assume that the cells you are using are adherent cells. You're walking a fine line here: your protein is secreted into the media. Therefore, when you change the media you're going to slightly shock your cells. And you don't want to have the issue of shocking your cells and having them output less of your secreted protein.
Is the issue having bovine proteins or specifically the Bovine Ig molecules? If its the former, you could consider using a synthetic serum (it costs less). If its the later, you could use a low IgG serum and culture your cells in say a 5% serum-containing medium.
The last option would be to use serum-free cells.
If those options aren't favorable, I would probably change the media earlier following transfection rather than later.

~Labrat612

[Benita]

Hey Labrat,

thx for your advices.

I try to purificate PSCA protein from the media. It's extracellular region is cloned into pSecTag2 expression Vector from Invitrogen. If purificate with nickel columns (FastFlow Ge Healthcare) including FCS in the media I have a lot of  contaminations next to my protein itself. I need a purity of up to 95%! So I decided to change the medium after transfection! I use HEK293T cells, adherent.

What kind of cells are serum free?

Thank you in advance.

Greetings,

Benita!

labrat612, on Jun 8 2010, 05:53 PM, said:

Benita, on Jun 8 2010, 05:50 AM, said:

Dear all,

I used to transfect my cells in 15cm diameter culture plates with 80% confluence using Fugene 6. My protein is secreted into the media so I have to change the media (from serum containing - [better growth an healthy cells] to serum free media [no fcs background proteins in my purification])  after transfection. So I'd like to ask what, in your opinion, is the best timepoint. Some people telling me "the next day" (so 12-18h) or others told me 4-6h after transfection.

I'm not sure. If I change it earlier maybe there is more protein in the medium because they live longer. But maybe the FCS is important for the first 12h of protein building? I don't know. What do you mean?

Greetings from Germany.

Franzi!

Hi Benita,

I'm going to assume that the cells you are using are adherent cells. You're walking a fine line here: your protein is secreted into the media. Therefore, when you change the media you're going to slightly shock your cells. And you don't want to have the issue of shocking your cells and having them output less of your secreted protein.
Is the issue having bovine proteins or specifically the Bovine Ig molecules? If its the former, you could consider using a synthetic serum (it costs less). If its the later, you could use a low IgG serum and culture your cells in say a 5% serum-containing medium.
The last option would be to use serum-free cells.
If those options aren't favorable, I would probably change the media earlier following transfection rather than later.

~Labrat612