Anti-Src Antibody Picoband®

Slit2/Robo1 signaling activates the Src-mediated inhibition of E-cadherin. IHC analysis of the expression of total Src and pSrc (Tyr416) in the tumor tissues of Apc Min/+ and Apc Min/+ ; Slit2 mice (A). Inactivation of Slit2/Robo1 significantly reduced the expression of pSrc (Tyr 416) in SW620 and SW480 cells, and activation of Slit2/Robo1 signaling through overexpressing Slit2 or Robo1 expression promotes pSrc (Tyr 416) expression in HCT-116 cells (B). IHC analysis of the expression of E-cadherin in the tumor tissues of the Apc Min/+ and Apc Min/+ ; Slit2 mice (C). Inactivation of Src signaling significantly enhances the expression of E-cadherin but inhibits the expression of β-cateninin in SW620 and SW480 cells (D). Src inactivation in SW620 and SW480 cells also led to a reduced nuclear translocation of β-catenin (E). Inhibition of E-cadherin expression through siRNA technique could promote the expression of β-cateninin and vimentin in SW620 and SW480 cells (F). IHC analysis of the expression of vimentin in the tumor tissues of Apc Min/+ and Apc Min/+ ; Slit2 mice (G). The data in IHC staining are representative of 11 mice per group (All mice were 24-week-old). The results of IHC were determined using IPP software, and the data were expressed as the mean ±S.D. *: P < 0.05, **: P < 0.01. Scale bars: 20 μm (A, C and G) and 25 μm (E).
Index in PubMed under a CC BY license. PMID: 25605242

Western blot analysis of Src using anti-Src antibody (PB9059).
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 30 ug of sample under reducing conditions.
Lane 1: human A549 whole cell lysates,
Lane 2: human RT4 whole cell lysates,
Lane 3: human PC-3 whole cell lysates,
Lane 4: human SH-SY5Y whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA 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-Src antigen affinity purified polyclonal antibody (Catalog # PB9059) 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 1:5000 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 Src at approximately 60 kDa. The expected band size for Src is at 60 kDa.

Flow Cytometry analysis of HepG2 cells using anti-Src antibody (PB9059).
Overlay histogram showing HepG2 cells stained with PB9059 (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-Src Antibody (PB9059, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

IHC analysis of Src using anti-Src antibody (PB9059).
Src was detected in a paraffin-embedded section of human liver cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-Src Antibody (PB9059) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IHC analysis of Src using anti-Src antibody (PB9059).
Src was detected in a paraffin-embedded section of human rectal cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-Src Antibody (PB9059) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.