Positive control (cell lysate), for antibodies to Asc (human)

Figure: Detection of full-length human Asc (aa 1-195) with PAb to Asc (human) (AL177) (Prod. No. ALX-210-905) in a cell lysate control of mock transfected cells (HEK 293T) (Prod. No. ALX-840-600) (lane 1) and HEK 293T cells transfected with a full-length human Asc expression plasmid (Prod. No. ALX-840-602) (lane 2). Note that overexpressed Asc migrates at ~30kDa while endogenous Asc migrates at ~23kDa.Method: 13µl of water and 2µl of DTT (100mM) were added to 5µl of cell lysate (diluted 10x Prod. No. ALX-840-602) (corresponding to 10⁴ cells) and boiled 5 min before being loaded on a 12% SDS-PAGE gel. The gel was run at 200V and proteins transferred to nitrocellulose (Amersham Biosciences, Prod. No. 25006565) for 1hour at 100V in transfer buffer (3g/l trizma-base, 14.4g/l glycin, 20% methanol at pH 8.3). The nitrocellulose membrane was then blocked in PBS-Tween 0.1%, milk 4% at 4°C and incubated with PAb to Asc (human) (AL177) (Prod. No. ALX-210-905) diluted 1:1’000 in PBS-Tween 0.1%, milk 4% for 1 hour at RT. After three washes in PBS-Tween 0.1%, the membrane was incubated with goat anti-rabbit HRP diluted 1:5’000 in PBS-Tween 0.1%, milk 4% for 45 min at RT. After three washes in PBS-Tween 0.1%, the membrane was revealed in ECL solution (Pierce, Prod. No. 34080). The membrane was exposed for 30 sec. with a Fujimedical X-Ray film (Fujifilm, Prod. No. 57164053).