Anti GRP78 BiP, Recombinant Antibody

Immunohistochemical analysis of paraffin-embedded human pancreas, using GRP78 BiP Antibody(M00955)HSPA5 was detected in paraffin-embedded tissue section. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml rabbit anti-HSPA5 Antibody (M00955)overnight at 4℃. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37℃. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

Western blot analysis of GRP78 BiP expression in (1) LnCaP cell lysate;(2) HepG2 cell lysate (M00955). 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. 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-HSPA5 monoclonal antibody (Catalog # M00955) overnight at 4℃, 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 HSPA5