Cleared RIPA lysates from transfected HEK293T cells were incubated with anti-FLAG M2 affinity gel beads (Sigma) for 4hrs at 4°C. Beads were washed in RIPA buffer 5 times, then PBS, and FLAG-Dvl1 was eluted with 100μL FLAG-peptide (Sigma) solution (0.5mg/mL peptide, PBS, protease and phosphatase inhibitors) for 30 minutes at 4°C. Beads were removed and a fraction of eluate was saved. Purified protein was re-suspended in 2mL blocking buffer (1x PBS-T (0.1% Tween-20), 3% BSA) with inhibitors, and added to blocked membrane lipid strips (Echelon) for 1hr at room temperature. Membranes were washed in PBS-T, treated with primary and secondary antibody, and developed. The amount of FLAG-Dvl1 bound to lipid was normalized to the amount eluted.
Blocked membrane lipid strips
Manufactured by Echelon Biosciences
Blocked membrane lipid strips are a laboratory tool used to detect and analyze specific lipid-binding interactions. These strips contain a variety of immobilized lipids and are commonly used in lipidomic and biochemical research applications.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using blocked membrane lipid strips
1
Probing Dvl1-Lipid Interactions
2
Probing Dvl1-Lipid Interactions
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Cleared RIPA lysates from transfected HEK293T cells were incubated with anti-FLAG M2 affinity gel beads (Sigma) for 4hrs at 4°C. Beads were washed in RIPA buffer 5 times, then PBS, and FLAG-Dvl1 was eluted with 100μL FLAG-peptide (Sigma) solution (0.5mg/mL peptide, PBS, protease and phosphatase inhibitors) for 30 minutes at 4°C. Beads were removed and a fraction of eluate was saved. Purified protein was re-suspended in 2mL blocking buffer (1x PBS-T (0.1% Tween-20), 3% BSA) with inhibitors, and added to blocked membrane lipid strips (Echelon) for 1hr at room temperature. Membranes were washed in PBS-T, treated with primary and secondary antibody, and developed. The amount of FLAG-Dvl1 bound to lipid was normalized to the amount eluted.
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