Product Page for SEMA4F antibody N-terminal region(ARP46421_P050)

Data Provided by Tianliang Sun, Max Planck Institute for Heart and Lung Research

Sample Description:

human cell line A431, load ab 500,000 cells per lane

How many different experimental trials were conducted using the antibody sample?

three

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

WB Procedure/Protocol:

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.