For recombinant protein immunoblotting, selected mAbs were used to probe 50, 10, 2, and 0.4 ng of recombinant protein antigens. For immunoblotting of endogenous human protein samples by PETAL mAbs, cell lines were selected according to the protein expression profile from HPA and UniProt databases. For membrane or nuclear proteins, corresponding cellular fractions were prepared for immunoblotting. Typically, 20 μg of protein was loaded onto each lane. Support-positive immunoblotting results were evaluated following the criteria described by Antibodypedia (http://antibodypedia.com/text/validation_criteria#western_blot) and HPA (29). Basically, an antibody was qualified as immunoblotting positive when the size of a single or predominant single band on immunoblotting matched or was within 10% of the predicted antigen molecular weight. In some cases, an immunoblotting-positive conclusion was enhanced when the same predicted protein band was detected in two or more different cell lysates. Some antibodies detected multiple bands with different sizes, but the predicted size protein band was also detected.

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.