Phosphoprotein immufluorescence protocol

Phosphoprotein immufluorescence protocol

Postby vald0drag » Wed Dec 14, 2011 3:10 pm

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.
vald0drag
 
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Re: phosphoprotein immufluorescence protocol

Postby geersc » Wed Dec 14, 2011 6:02 pm

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
geersc
 
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Re: phosphoprotein immufluorescence protocol

Postby vald0drag » Thu Dec 15, 2011 2:16 pm

Thank you! Will try that next week. I hope it will work on my cells too
vald0drag
 
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Joined: Fri Oct 21, 2011 10:21 am


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