Immunofluorescence (IF) (Fluorescent image of U251 cells stained with PI3KC3 (S34) antibody. U251 cells were treated with Chloroquine (50 uM,16h), then fixed with 4% PFA (20 min), permeabilized with Triton X-100 (0.2%, 30 min). Cells were then incubated with MBS9204783 PI3KC3 (S34) primary antibody (1:200, 2 h at room temperature). For secondary antibody, Alexa Fluor 488 conjugated donkey anti-rabbit antibody (green) was used (1:1000, 1h). Nuclei were counterstained with Hoechst 33342 (blue) (10 ug/ml, 5 min). PI3KC3 immunoreactivity is localized to autophagic vacuoles in the cytoplasm of U251 cells.)
Western Blot (WB) (Western blot analysis of PI3KC3 (S34) in Hela cell line lysates (35ug/lane). PI3KC3 (arrow) was detected using the purified Pab.)
Immunohistochemistry (IHC) (Formalin-fixed and paraffin-embedded human skeletal muscle tissue reacted with PI3KC3 Antibody (S34), which was peroxidase-conjugated to the secondary antibody, followed by DAB staining. This data demonstrates the use of this antibody for immunohistochemistry; clinical relevance has not been evaluated.)
Immunohistochemistry (IHC) (Formalin-fixed and paraffin-embedded H.liver tissue reacted with PI3KC3 Antibody (S34) (Cat#MBS9204783).)
Western Blot (WB) (Western blot analysis of PI 3 Kinase Class 3 expression in 293T cell lysate (MBS179834).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-PIK3C3 monoclonal antibody overnight at 4 degree 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 with Tanon 5200 system. A specific band was detected for PIK3C3 )
Immunohistochemistry (IHC) (Immunohistochemical analysis of paraffin-embedded human kidney, using PI 3 Kinase Class 3 Antibody(MBS179834)PIK3C3 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-PIK3C3 Antibody (MBS179834)overnight at 4 degree C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen. )
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