For #1, we take 100 ul lysis buffer that will serve as the control? Will we only have 1 “control” to refer to? Where did the #s come from in formula?

We took 50 ul lysis buffer as one control, two replicates, total 100 ul.

We referred to these two articles, titled Spectrophotometric determination of serum iron at the submicrogram level with a new reagent (ferrozine) and High-throughput non-heme iron assay for animal tissues). We got the protocol from Dr. Tracey Rouault group. See the two DOI below.

DOI: 10.1016/0003-2697(71)90405-2; DOI: 10.1016/j.jbbm.2004.01.001