Figure 1
Antibody screening and NCI-60 'reverse-phase' protein lysate microarrays. A) Sixteen 4-mm-wide nitrocellulose strips from a nitrocellulose membrane blot, each used to test a different antibody. Each blot was classified by the criteria described in the text. Asterisks indicate predominant band(s). Eight blots (53BP2, Brm, Btf, hCNK1, PTEN, Smad4, TIF2, and XPF) were classified as single band and the expected molecular weight. Four blots (HIF-1 α, IKK β, IKK γ, and pan-JNK) were classified as multiple bands. Two blots (CD54, and JNK pY185) were classified as wrong molecular weight. One blot (IGFBP-3) was classified as no band. All band results from this Western blotting were entered into AbMiner. It should be noted that the results in this figure may not correspond to the band results in AbMiner because the latter are sometimes updated after additional testing. B) Miniature incubation chambers for 4-mm strips used for incubations with primary and then secondary antibodies. C) Reverse-phase lysate arrays. Each row consists of 10 two-fold dilutions of an NCI-60 cell line or a control pool consisting of all 60 cell lines. Concentrated pool was spotted at the bottom-right corner of each field to serve as a registration mark for image processing. i) lysate array stained for total protein with SYPRO Ruby. ii) lysate array incubated with an antibody for p300, a protein ubiquitously expressed in the NCI-60. iii) CDK2 expression across 60 cell lines. Only one cell line shows visible expression (arrow). Nonetheless, expression of CDK2 was still detected as a single band by Western blot iv) Negative control. The primary antibody was replaced by anti-Aspergillus niger glucose oxidase IgG1, which does not recognize any human antigen.