1. Protocol for ELISA Sample Submission-384 well: General Procedures Primary assays: x-40 & x-42. (1-40, 1-42, & 1-total on special request). Linear range: 1pM (4pg/ml) - 110pM (500pg/ml). 50uL/well is needed for each assay. If both x-40 and x-42, submit 90uL. 2G3 and 21F12 capture antibodies used for x-40 and x-42 assays, respectively; 266B antibody used for detection. Submit samples on 96 well plate!! Please DO NOT use tissue culture plates. Please leave 1 row empty for peptide standard, and 1 column empty. Always submit a submission sheet ahead of time. Even if you talk to Ting about preparing a submission, you should submit a sheet at that time. This is the ONLY way that Ting can plan ahead and prepare for assays. Submission sheets can be emailed to Ting (tyang3@partners.org) or dropped off at Ting’s desk (Harvard Institute of Medicine, Xia Lab, HIM 610).

2. Your sample layout (on 96 well plate): --For 1 assay submit 50ul/sample; for 2 assays(x-40 & x-42) submit 90ul/well --Add samples to a 96 well plate, starting at B1 and moving across, as shown on left below. --Fill all 11 wells in each row before starting another row. --Fill up to 7 rows. Protocol for ELISA Sample Submission-duplicate: sample layout 1 2 3 4 5 6 7 8 9 10 11 12 ABCDEFGHABCDEFGH

3. Example of sample submission sheet 384 well duplicate