Whole cell extracts were extracted using RIPA and protein concentrations were determined by BCA assay. Equal aliquots of 50 μg total proteins were separated by SDS‐PAGE. Then proteins were transferred to a PVDF membrane. Membranes were blocked with 5% bovine serum albumin in TBST and then incubated in primary antibodies overnight at 4°C followed by secondary antibodies for 1 h at 37°C. Rabbit polyclonal primary antibody against CXCR4 (sc‐9046), mouse monoclonal antibodies against RhoA (sc‐418), RhoGEF (sc‐166301), ROCK (sc‐17794), PI3K (sc‐166365), PAK (sc‐166174), PKN (sc‐136037), GAPDH (sc‐166545), and the horseradish peroxidase‐conjugated secondary antibodies against rabbit‐IgG (sc‐2004) and mouse‐IgG (sc‐2005) were purchased from Santa Cruz Biotechnology. The primary rabbit polyclonal antibody against ARHGAP5 was purchased from Sigma‐Aldrich. Finally, signal was visualized through a chemiluminescent detection system (Pierce ECL Substrate Western blot detection system, Thermo, Rockford, IL, USA).
Horseradish peroxidase conjugated secondary antibodies against rabbit igg
Manufactured by Santa Cruz Biotechnology
Sourced in China
Horseradish peroxidase‐conjugated secondary antibodies against rabbit‐IgG. These antibodies are designed to bind to rabbit immunoglobulin G (IgG) and are conjugated with the enzyme horseradish peroxidase.
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Lab products found in correlation
2 protocols using horseradish peroxidase conjugated secondary antibodies against rabbit igg
1
Quantitative Western Blot Analysis
2
Western Blotting Protein Extraction
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Protein extraction was performed using the Western blotting technique (Shah et al., 2020b (link)). Proteins were loaded on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE, 12%) to separate and shifted to nitrocellulose membranes. After being blocked with 5% non-fat milk in TBST for 2 h and being incubated with diluted primary chicken antibodies including LC3 (1:500), Beclin-1 (1:1,000), PTEN (1:1,000), AKT (1:500), β-actin (1:1,000), mTOR (1:500) (ImmonuWay), and ACO-2, HK-1 and SDHB (1:1,000) at 1 h for 37°C, then the membranes were washed 4 times with PBST for 5 min each time and were incubated in horseradish peroxidase–conjugated secondary antibodies against rabbit IgG (1:2000; Santa Cruz, CA) at 37°C for 1 h. Enhanced chemiluminescence (ELC Biosharp Life Sciences, Anhui, China) and image J software (National Institute of Health, Bethesda, MD) were used for the visuals of bound immune complexes.
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