Data Provided by Tianliang Sun, Max Planck Institute for Heart and Lung Research
human cell line A431, load ab 500,000 cells per lane
How many different experimental trials were conducted using the antibody sample?
How was this sample prepared?
Transfection control siRNA or sema4F siRNA to cells, after 48 hours lysate the cell for WB.
6well plate full of cells, add 200 ul RIPA buffer per well, 4 degree 20 min rotation then centrifuge 10min at maxi rmp. Pick the supernatant and boiled 5min with lamili.
Primary antibody dilution and incubation time:1:600, 4 degree overnight
Secondary antibody used and dilution and incubation time: 1:3000, RT 2 hours
What controls were used in your experiment (positive/negative)?
Control siRNA knockdown
Samples are load on 8% SDS PAGE gel, 40ul per well.
150v for 1.5 hours
Transfer the gel to membrane (10%methal) at 400mA ,2hours.
Block with 5% milk in TBST 1hour at RT
Incubate with antibody diluted in block buffer at 4 degree overnight
Wash 3 time with TBST , 15min each
2nd antibody dilute in block buffer at RT for 2 hours
Wash 3 times with TBST; 15 min each
Develop with ECL in dark room.