Animal cell protein extraction kit

Roche's animal cell protein extraction kit was used to extract the total protein in the sample (specific operation according to the specification) and the operation was optimized (12 (link)). Rabbit monoclonal TAG1 antibody (dilution, 1:500; cat. no. ab133498) and rabbit monoclonal APP antibody (dilution, 1:500; cat. no. ab180140) were purchased from Abcam (Cambridge, MA, USA).

The animal cell protein extraction kit (Roche Diagnostics) was used to extract the total proteins. Western blot analysis was performed as described previously (13 (link)). After protein quantitation using the Lowery protein assay, equal amounts of proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes by the semi-dry blotting method using a three-buffer system. The membranes were incubated with primary antibody at a dilution of 1:500 (anti-CDX2, RayBiotech, Inc., Norcross, GA, USA; cat no.: 119-14329) overnight at 4°C. The membrane was washed with TBST and incubated with a peroxidase-conjugated secondary polyclonal goat-anti-mouse antibody (1:1000) (Santa Cruz Biotechnology, Inc. CA, USA; cat no.: sc-395763) for 1 h. Western blot film was scanned and the membrane was stripped and reprobed with antibody against β-actin (1:1500) (Santa Cruz Biotechnology, Inc.; cat no.: sc-8432) to confirm equal sample loading