Hi everyone,
I'm trying to stain FLAG-tagged membrane protein on Fibroblast cell line. The protocol given to me is so simple but I wonder why I cannot see the localization of the protein.. I used single antibody which is anti-FLAG-FITC. I'm sure tranfection was successful because I used DS-Red as a marker, and I checked before I fixed the cell. The protocols are: 1. fixed with 4% PFA (15min) 2. permeabilize with 0.5% triton x-100 (15min).. 3. block with 1% BSA (1 hr) 4. tag with antibody (1 hr) 5. Mount. Can someone pls give some ideas what my problem might be? tq in adv..
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