The cross-linker dithiobis(succinimidyl propionate) (DSP; Thermo Fisher Scientific no. 22586) was freshly prepared at 20 mM in dimethyl sulfoxide and diluted to a working concentration of 0.5 mM in PBS. Cells were washed twice with PBS and incubated in 0.5 mM DSP for 30 min at room temperature. After removal of DSP, cells were incubated with quenching solution [20 mM tris-Cl (pH 7.4)] for 15 min at room temperature, lysed, and incubated with anti-TRAPα antibodies for 6 hours at 4°C. Immunoprecipitates were washed, subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-TRAPα and anti-proinsulin antibodies.

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

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.