Western blotting

Detection of HA-hM3Dq using western blot

To assess HA-tagged hM3Dq DREADD expression in the hippocampus and cortex of CamKIIα-tTA::TetO-hM3Dq bigenic mice on postnatal day 7 (P7), we performed western blotting analysis for the HA antigen.

Tissue samples were dissected and stored at -80 °C, and then homogenized in Radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) using a Dounce homogenizer. The lysis buffer contained protease and phosphatase inhibitors (Sigma Aldrich, United States). 

Following the estimation of protein concentration using the Quantipro BCA assay kit (Sigma-Alrich, United States), equal amounts of lysate (40 ug) were resolved on a 10% sodium dodecyl sulfate polyacrylamide gel and then transferred onto polyvinylidene fluoride membranes (100V, 80-90 minutes, wet transfer). 

Blots were blocked in 5% milk dissolved in TBST for one hour, and subsequently incubated overnight with rabbit anti-HA antibody (1:1500 in 5% milk, Cat. No. H6908, Sigma-Aldrich, United States). Following subsequent washes with TBST, blots were exposed to HRP conjugated goat anti-rabbit secondary antibody (1:6000, Cat. No. AS014, Abclonal Technology, United States) for one hour. Signal was visualized using a GE Amersham Imager 600 (GE life sciences) with a western blotting detection kit (WesternBright ECL, Advansta, United States). Densitometric quantitative analysis was performed using ImageJ software.

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