Alkaline phosphatase conjugated goat anti mouse igg antibody

The protein of anterior lens capsules and lens cortex was extracted in lysis buffer (1 M Tris-HCl at pH 7.5, 1% Triton X-100, 1% Nonidet p-40, 10% SDS, 0.5% sodium deoxycholate, 0.5 M EDTA, 10 mg/mL leupeptin, 10 mg/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Proteins were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride filter membranes (Millipore, Bedford, MA). Nonspecific protein binding to the membrane was blocked with blocking buffer (5% nonfat milk, 200 mM NaCl, 50 mM Tris, and 0.05% Tween 20). The blocked membrane was then incubated with mouse anti-human-WRN (1 : 800; Abcam, Cambridge, UK) and anti-GAPDH (1 : 1000; Santa Cruz, CA, USA) at 4°C for 18 h. The membrane was washed three times with TBST (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20) for 5 min each time, followed by incubating with an alkaline phosphatase-conjugated goat anti-mouse IgG antibody (1 : 2000; Santa Cruz, CA, USA) for 2 h. Detection was performed using an ECL chemiluminescence kit (Pierce, Rockford, IL) and the signal was exposed to an X-ray film that was scanned using Image Quant software (Molecular Dynamics, Sunnyvale, CA, USA).

The protein of LECs and HLEPIC were extracted separately in lysis buffer (1 M Tris-HCl at pH 7.5, 1% Triton X-100,1% Nonidet p-40, 10% SDS, 0.5% sodium deoxycholate, 0.5 M EDTA, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride [PMSF]). Equal amounts of proteins were size fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 15% polyacrylamide gels. Proteins were then transferred onto polyvinylidene difluoride filter membranes (Millipore, Bedford, MA, USA). The blocked membrane was then incubated with mouse anti-human-ATM (Sigma) and mouse antihuman-GAPDH (1:1000; Abcam, Cambridge, UK) at 4 °C for 12 hours. After washing, the membrane was incubated with an alkaline phosphatase-conjugated goat anti-mouse IgG antibody (1:2000; Santa Cruz) for 2 hours. An enhanced chemiluminescence detection system was used to read the Western signals (Pierce Company, USA).