Figure: Detection of full-length human NLRP1/NALP1 (aa 1-1429) with MAb to NLRP1/NALP1 (human) (Nalpy1-4) (Prod. No.
ALX-804-803) 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 NLRP1/NALP1 expression plasmid (Prod. No.
ALX-840-603) (lane 2).
Method: 13µl of water and 2µl of DTT (100mM) were added to 5µl of cell lysate (diluted 10x Prod. No.
ALX-840-603) (corresponding to 10
5 cells) and boiled 5 min before being loaded on a 8% SDS-PAGE gel. The gel was run at 200V and proteins transferred to nitrocellulose (Amersham Biosciences, Prod. No. 25006565) first for 30 min at 100V in transfer buffer (3g/l trizma-base, 14.4g/l glycin, 20% methanol at pH 8.3) containing 0.1% SDS and then for 45 min. in normal transfer buffer. The nitrocellulose membrane was then blocked in PBS-Tween 0.1%, milk 4% at 4°C and incubated with MAb to NLRP1/NALP1 (human) (Nalpy1-4) (Prod. No.
ALX-804-803) at 1µg/ml 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 PAb to mouse IgG1 (Prod. No.
ALX-211-203) 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 5 min. with Fujimedical X-Ray film (Fujifilm, Prod. No. 57164053)."