Anti-DR4/TNFRSF10A Antibody Picoband®

Western blot analysis of DR4 using anti-DR4 antibody (A02152).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
lane 1: rat spleen tissue lysates,
lane 2: mouse spleen tissue lysates,
lane 3: MCF-7 whole cell lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-DR4 antigen affinity purified polyclonal antibody (Catalog # A02152) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for DR4 at approximately 50KD. The expected band size for DR4 is at 50KD.

IF analysis of ATG14L using anti-ATG14L antibody (A02152).
ATG14L was detected in an immunocytochemical section of U2OS cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-ATG14L Antibody (A02152) overnight at 4°C. Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

The cytotoxic effect of TRAIL on human HCC and GC cell lines. (A) Detection of DNA fragmentation by DNA ladder assay. LH86, Huh7, HLCZ01 and HLCZ02 cells were exposed to TRAIL for 4 hours. 2 μg of cellular DNA was separated on 1% agarose gel at 50V for one hour. The data are one representative of three independent experiments. (B) Detection of apoptosis by flow cytometry and western blot. LH86, Huh7, HLCZ01, HLCZ02, HGC-27 and BGC-823 cells were exposed to TRAIL for 4 hours. Samples were analyzed on a FACS Caliber Cytometer. A minimum of 30000 events per samples were acquired, and subsequently analyzed with CellQuest software. The results are the average of at least three independent experiments. (C) Cleaved PARP was detected by western blot. β–actin was used as control. The data are one representative of three independent experiments. (D) Detection of DR4 and DR5 in HCC and GC cell lines by real-time PCR and western blot analysis. DR4 or DR5 mRNA were detected by real-time RT-PCR and normalized with GAPDH respectively. The results are the average of three independent experiments performed in triplicate. DR4 and DR5 protein was detected by western blot. The data are one representative of three independent experiments.
Index in PubMed under a CC BY license. PMID: 24970806