Anti human gapdh sc 25778

Whole endometrial tissue lysate was prepared and homogenized in lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, and 0.25% sodium deoxycholate). Protein concentration was measured and adjusted by using the BCA Reagent kit (Applygen, Beijing, China). Samples were run on a 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking in 5% nonfat dry milk in TBST (0.1% Tween 20 in TBS) for 1 h, membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-human telomerase (ab68781, Abcam, Cambridge, UK), ER alpha (sc-7207, Santa Cruz, CA, USA) or anti-human Gapdh (sc-25778, Santa Cruz). After three washes in TBST, membranes were incubated with secondary antibodies conjugated with horseradish perioxidase for 1 h at about 25°C. The signals were developed with the ECL Chemiluminescent kit (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). Films were scanned using a flat-bed scanner. The intensities of the bands representing ER alpha and TERT were evaluated using the Image J analysis software (Rawak Software, Inc., Germany).