detecting fluorescence - (Jan/08/2007 )
I've made stable cell lines of HEK293 cells transfected with my protein of interest fused with red fluorescent protein.My protein is secreted on the cell surface.By doing western i've confirmed that along with normal protein,protein fused with RFP is also secreted by cells.Now i want to check fluorescence in media at different time intervals.
I tried it using fluorescence spectrophotometer but couldn't detect anything,not even in cell lysis.
Can anyone suggest me what should i do?
Someone suggested me run native gel & then check fluorescence in it.Does it work?
Please help...
-minnee-
QUOTE (minnee @ Jan 8 2007, 06:32 PM)
I've made stable cell lines of HEK293 cells transfected with my protein of interest fused with red fluorescent protein.My protein is secreted on the cell surface.By doing western i've confirmed that along with normal protein,protein fused with RFP is also secreted by cells.Now i want to check fluorescence in media at different time intervals.
I tried it using fluorescence spectrophotometer but couldn't detect anything,not even in cell lysis.
Can anyone suggest me what should i do?
Someone suggested me run native gel & then check fluorescence in it.Does it work?
Please help...
I tried it using fluorescence spectrophotometer but couldn't detect anything,not even in cell lysis.
Can anyone suggest me what should i do?
Someone suggested me run native gel & then check fluorescence in it.Does it work?
Please help...
I think I once have read that fluorescent proteins need some trace elements for fluorescence; moreover, FP are sensitive for oxidation; may be secreted FP are too diluted or quenched by medium which abolosh fluorescence
-The Bearer-
QUOTE (kosmodrom @ Jan 8 2007, 11:57 AM)
QUOTE (minnee @ Jan 8 2007, 06:32 PM)
I've made stable cell lines of HEK293 cells transfected with my protein of interest fused with red fluorescent protein.My protein is secreted on the cell surface.By doing western i've confirmed that along with normal protein,protein fused with RFP is also secreted by cells.Now i want to check fluorescence in media at different time intervals.
I tried it using fluorescence spectrophotometer but couldn't detect anything,not even in cell lysis.
Can anyone suggest me what should i do?
Someone suggested me run native gel & then check fluorescence in it.Does it work?
Please help...
I tried it using fluorescence spectrophotometer but couldn't detect anything,not even in cell lysis.
Can anyone suggest me what should i do?
Someone suggested me run native gel & then check fluorescence in it.Does it work?
Please help...
I think I once have read that fluorescent proteins need some trace elements for fluorescence; moreover, FP are sensitive for oxidation; may be secreted FP are too diluted or quenched by medium which abolosh fluorescence
what is the color of the medium ? red ???
-beta_hong-
what is the color of the medium ? red ???
[/quote]
Not exactly red.It is orangish red.
-minnee-