Phosphoprotein immufluorescence protocol

[vald0drag]

Has anyone of you stained cells with phosphospecific antibodies with red or green fluorescence? I would like a working protocol. My cells are MDCKs and 293Ts... In MDCK we want to detect endogenous phosphoprotein, in 293T - transfected.

[geersc]

Hi vald0drag, i stained mpk-CCD Cells with phosphospecific antibodies with green and red fluorescence.
First we wash the cells 3 times for 5 minutes with TBS
Then fixation for 10 min with 4 % PFA in TBS
wash (3x 5 min TBS)
Then treatment with 0,1 % Triton in TBS for 10 min
wash (3x 5 min TBS)
Blocking for 1 hr in 5 % skim milk in TBS
Primary antibody incubation in 5 % skim milk in TBS 1 hr at room temp and then overnight at 4 °C
wash (3x 5 min TBS)
Secondary antibody incubation in 5 % skim milk in TBS for 1,5 hr (red/CY3 1:250; green/CY2 1:50)
wash (3x 5 min TBS)
if required: DAPI 1 staining 5 min in TBS and wash 1x 5 min
I used the prolong antifade kit as a mounting medium for the cells
With that protocoll i was able to take some nice pictures on the confocal microscope

I hope this will help you and i wish to you good luck with your experiments

[vald0drag]

Thank you! Will try that next week. I hope it will work on my cells too