Hrp conjugated affinipure goat anti rabbit igg

Total cellular protein was extracted with RIPA lysate buffer (Boster Biological Technology) containing protease inhibitor and phosphorylase inhibitor (Boster Biological Technology), and determined protein concentration with BCA protein detection kit (Boster Biological Technology), then separated by SDS-PAGE electrophoresis kit (cwbiotech). The amount of each group proteins was 20μg. After separation, the proteins were transferred to PVDF membrane (0.22um). When sealed by 0.5% non-fat milk powder still 1 h, the PVDF membrane was incubated with polyclonal primary antibody and secondary antibody combined with HRP. The membrane was colored by ECL enhanced chemiluminescence kit (Boster Biological Technology) and formed by Biological Spectrum Image System Scanning.
Primary antibodies used in this study include anti-β-actin(1:5000, AP0060, Bioworld Technology, Inc), anti-bcl-2(1:1000, AF6139, Affinity), anti-Bax(1:1000, AF0120, Affinity), anti-p-VEGFR-2(1:500, AF3279, Affinity), anti-MMP-2(1:500, AF5330, Affinity). The secondary antibodies include HRP-conjugated affinipure goat anti-Rabbit IgG(1:5000, Boster Biological Technology, BA1054).

Total proteins were prepared using TRIzol reagent following the protocol. Protein concentration was determined using a BCA Protein Assay Kit (P0010; Beyotime). Protein extracts were denatured in sample loading buffer and resolved by 12% SDS‐PAGE gel, and then the proteins were migrated from the gel onto the PVDF membrane (Immun‐BlotTM PVDF Membrane, Bio‐Rad) which was pre‐soaked in methanol and transfer buffer. The membranes were blocked with 5% nonfat dried milk in PBS for 1 h at room temperature and then incubated in blocking solution at 4°C overnight with primary antibodies, which included rabbit anti‐PI3K polyclonal antibody(Ab182651; BACAM, 1:500 dilution), rabbit anti‐AKT1 monoclonal antibody (Ab81283; BACAM, 1:1000 dilution) and rabbit anti‐S100 alpha 6 monoclonal antibody (Ab181975; BACAM, 1:1000 dilution). Membranes were incubated with a secondary antibody, which was HRP Conjugated AffiniPure goat Anti‐rabbit IgG (BA1054; Boster Biological Technology, Ltd., 1:5000 dilution) in blocking solution at room temperature for 1 h. Chemiluminescence was used to detect the target protein, the membranes were covered with substrate solution and incubated. The immunoreactive bands were quantified by using Image J software and GADPH was used as internal reference protein for quantitative analysis.