Anti-Integrin beta 5/ITGB5 Antibody Picoband®

Western blot analysis of Integrin beta 5/ITGB5 using anti-Integrin beta 5/ITGB5 antibody (A04201-1).
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: human A549 whole cell lysates,
Lane 2: mouse Ana-1 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-Integrin beta 5/ITGB5 antigen affinity purified polyclonal antibody (Catalog # A04201-1) 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 Integrin beta 5/ITGB5 at approximately 90KD. The expected band size for Integrin beta 5/ITGB5 is at 90KD.

IHC analysis of ITGB5 using anti-ITGB5 antibody (A04201-1).
ITGB5 was detected in paraffin-embedded section of human rectal cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-ITGB5 Antibody (A04201-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

IF analysis of Integrin beta 5/ITGB5 using anti-Integrin beta 5/ITGB5 antibody (A04201-1).
Integrin beta 5/ITGB5 was detected in immunocytochemical section of A431 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 4μg/mL rabbit anti-Integrin beta 5/ITGB5 Antibody (A04201-1) overnight at 4°C. DyLight®550 Conjugated Goat Anti-Rabbit IgG (BA1135) was used as secondary antibody at 1:100 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.

Flow Cytometry analysis of A549 cells using anti-Integrin beta 5/ITGB5 antibody (A04201-1).
Overlay histogram showing A549 cells stained with A04201-1 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Integrin beta 5/ITGB5 Antibody (A04201-1,1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Flow Cytometry analysis of Raji cells using anti-Integrin beta 5/ITGB5 antibody (A04201-1).
Overlay histogram showing Raji cells stained with A04201-1 (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-Integrin beta 5/ITGB5 Antibody (A04201-1,1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Western blot validation of proteomic data. ( A ) Elevated levels of ITGB5, TNXB , CA II, CA III were observed in the peri-implant sclerosis samples, while elevated levels of ACP5 and CTSK were observed in femoral head necrosis samples. ( B ) The ratio of TIGAR/GAPDH intensities in Western blot. GAPDH was used as a loading control, quantified and normalized to GAPDH using ImageJ. All experiments were performed in triplicate, * indicates significant expression level change compared to the control group, *P < 0.05, **P < 0.01. ***P < 0.001.
Index in PubMed under a CC BY license. PMID: 38851808