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. Author manuscript; available in PMC: 2012 Nov 1.
Published in final edited form as: J Pharmacol Toxicol Methods. 2011 Jun 30;64(3):233–237. doi: 10.1016/j.vascn.2011.06.001

Figure 1.

Figure 1

Time course of cell lysate-primary antibody binding in nitrocellulose membranes.

Nitrocellulose membrane were dotted with OVCAR-3 ovarian cancer cell lysates at 25 μg per 5 μL, air-dried, wetted with distilled water, and blocked with 5% milk for 1 hour. The membrane pieces were washed, dried, and wetted with water before incubation with 250 ng/mL Bad, cMyc, GAPDH, or HIF-1β primary antibodies in 5% milk with shaking for varying time at room temperature. These membrane pieces were then washed and incubated with 250 ng/mL Goat-anti-mouse-poly-HRP in 5% milk overnight. The membrane pieces were washed and detected with SuperSignal West Pico Chemiluminescent Substrate and x-ray films. Shown above is the image of cell lysate dots incubated with different primary antibodies for varying times. Dot values were quantitated with NIH ImageJ software and the 48-hr and 0-min incubation dots were set as 100% and 0%, respectively, for normalization and plotting.