Anti goat horseradish peroxidase conjugate secondary antibodies

Cells lines were washed with PBS and lysed at 0°C for 30 min using lysis buffer (120 mM NaCl, 30 mM KCl, 0.1% DTT, 0.5% Triton X-100) supplemented with protease and phosphatase inhibitor cocktail (Halt Protease Inhibitor Cocktail/ Halt Phosphatase Inhibitor Cocktail, Thermo Fisher Scientific Inc.). Cell lysate was sonicated at 4°C for 10 sec and subsequently centrifuged at 15 000x g for 20 min. Supernatant was carefully removed and protein content was measured by the Bradford method (Bio-Rad, Hercules, CA); and the supernatants were stored at 80°C.
Equal amounts of proteins extracts were separated on a 4–15% SDS PAGE precast gel (Bio-Rad), and transferred to a nitrocellulose membrane followed by immunoblotting.
Rabbit monoclonal antibody against BRCA1 (clone D-20, Santa Cruz) was used at a final concentration of 1μg/ml. Goat monoclonal antibody against gelsolin (clone C-20, Santa Cruz) was used at a final concentration of 1μg/ml.
HRP-conjugated γ-Tubulin (clone C-20, Santa Cruz) was used at a final concentration of 1μg/ml to ensure equal amount of protein loading.
The signal was detected using anti-goat horseradish peroxidase-conjugate secondary antibodies, and developed by enhanced chemiluminescence (Santa Cruz)

Western blot analysis was done to verify the expression of gelsolin in cancer and healthy carrier patients compared to healthy control.
Equal amounts of plasma proteins (50μg/lane) were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad).
After addition of the blocking mixture, the membranes were incubated with anti-gelsolin goat monoclonal antibody (clone C-20 Santa-Cruz) at appropriate dilutions at 4°C for 2 h. The signal was detected using anti-goat horseradish peroxidase-conjugate secondary antibodies, and ECL (Santa Cruz).
Serum protein concentration for each sample was measured in triplicate using the dye-binding protein (Bio-Rad) with human serum albumin as standard curve. To ensure uniform gel loading, the membranes were stripped for 30 min at 50 C in 62.5 mm Tris-HCl (pH 6.8), 2% SDS, and 100 mm β-mercaptoethanol, blocked in 2% BSA, and reprobed with specific γ-tubulin horseradish peroxidase-conjugate primary antibodies, and ECL (Santa Cruz) accordingly to Seonyoung C. et al. [28 (link)–29 (link)].