I am trying to look for clathrin co-localization with some membrane proteins while they are still on the membrane and before endocytosis. I am using the X22 mouse anti-clathrin monoclonal antibody. Has anyone seen co-localization of clathrin with tight junctions, gap junctions, or GPCRs on the plasma membrane? If so, what protocol did you use to make this work? I am having difficulty finding the correct protocol. I have used formaldehyde/triton and methanol fixations with no luck.
The problem is that methanol or triton dissolve the cell membranes. Even after formaldehyde fixation the proteins will be removed. Below is a procedure we use. Medium 1 is a HEPES-buffered saline.
Wash cells 3x with medium 1.
Fix cells with 3% paraformaldehyde in PBS, pH 7.4, 5-10 minutes at room temp.
Wash cells 3x with medium 1.
Permeabilize cells with 0.025% saponin (w/v) in medium 1 containing 0.5% bovine serum albumin, to block non specific antibody binding (AB buffer).
Incubate cells with primary antibody in AB buffer for 45-60 minutes at room temperature (determine amount of antibody to use by titration but a good starting point is approximately 5 ug/ml).
Wash cells 3x with Medium 1.
Incubate cells with secondary antibody goat anti-rabbit or mouse IgG conjugated to fluorophore in AB buffer for 30 minutes at room temperature. (working range for Molecular Probes Ab-conjugates is ~1:200-1:1000).
Wash cells 3x with medium 1.
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