Quantitative Western blotting for the AIM number associating with a pentameric IgM

HEK293T cells transfected with pCAGGS-AIM-HA were cocultured with HEK293T cells transfected with both pCAGGS-FLAG-mouse Fc and pCAGGS-mouse J chain–HA in DMEM, supplemented with 10% FBS for 1 day. From the culture supernatant, the Fc-pentamer/J-HA/AIM-HA complex was purified using the ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich), followed by a rabbit anti-AIM polyclonal antibody (rab2, made in house) conjugated to a HiTrap NHS-activated HP column (GE Healthcare Life Sciences, PA). The eluate was analyzed for J chain and AIM by Western blotting in a reducing condition using an anti-HA antibody. The intensity of the signal was measured using the NIH ImageJ software.

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.