Li-Cor WB optimisation - WB background at y680 of Li-Cor (Jun/02/2005 )
Hi all,
I am new to WB's and new to using the Li-Cor Odyssey so working out which step to alter next is tricky.
The bands produced by my protein of interest when doing the Li-Cor procedure are not as pronounced as when doing the usual colourimetric WB. However, I also have too much background at the 680 wavelength (red).
Should I fiddle with blocking buffers (have tried Li-cor Blocking buffer:PBS and 1% casein in PBS) or just try increasing my primary antibody concentration and decrease the secondary antibody concentration?
I am currently blocking for 2-3 hours at RT.
Hope you can help.
-lamcam-
QUOTE (lamcam @ Jun 2 2005, 05:25 PM)
Hi all,
I am new to WB's and new to using the Li-Cor Odyssey so working out which step to alter next is tricky.
The bands produced by my protein of interest when doing the Li-Cor procedure are not as pronounced as when doing the usual colourimetric WB. However, I also have too much background at the 680 wavelength (red).
Should I fiddle with blocking buffers (have tried Li-cor Blocking buffer:PBS and 1% casein in PBS) or just try increasing my primary antibody concentration and decrease the secondary antibody concentration?
I am currently blocking for 2-3 hours at RT.
Hope you can help.
I am new to WB's and new to using the Li-Cor Odyssey so working out which step to alter next is tricky.
The bands produced by my protein of interest when doing the Li-Cor procedure are not as pronounced as when doing the usual colourimetric WB. However, I also have too much background at the 680 wavelength (red).
Should I fiddle with blocking buffers (have tried Li-cor Blocking buffer:PBS and 1% casein in PBS) or just try increasing my primary antibody concentration and decrease the secondary antibody concentration?
I am currently blocking for 2-3 hours at RT.
Hope you can help.
Hi, I generally don't have much background on the 680 channel but the 800 channel does give me some background. I block for 1 hour in 5% milk in TBS and I dilute the secondary antibody 1:10,000 in 5% milk in TBS-T. I incubate with secondary for 1 hour. I am assuming that by "background" you mean the red haze that comes up and not other bands that you weren't expecting to see.
-Dynein-