Total protein was extracted using RIPA lysis buffer (cat.no. R0020, Solarbio Technology, Beijing, China) containing Phosphatase Inhibitor and Protease Inhibitor Cocktail (cat.no. C0001,MCE HY-K0023, Targetmol, MA, USA) at 4°C. About 10 µg of total protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane (cat.no. ISEQ00010, Merck KGaA, Darmstadt, Germany). After blocking in the blocking buffer containing 5% skimmed milk powder, the membranes were incubated with the rabbit anti-human KDF1 antibody (cat.no. PA5-55926, Thermo Fisher Scientific, MA, USA, dilution 1:1000) or mice anti-human GAPDH antibody (cat.no. YM3029, Immunoway, TX,USA, dilution 1:1000), overnight at 4°C. Then the membrane was washed and incubated with HRP-conjugated goat anti-rabbit (cat.no. B0201, Immunoway, TX, USA) or rabbit anti-mouse (cat.no. B0101, Immunoway, TX, USA) antibody for 2 h at 37°C. The immunolabeled proteins were detected by chemiluminescence using the Chemiluminescent hRP substrate (Merck KGaA, Darmstadt, Germany). Densitometric analysis was performed using the 1.52a version Image J software (National Institutes of Health, MD, USA).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.