Protocol for Printing Slides and Antibody Labeling
Cell or tissue lysates are arrayed on nitrocellulose-coated slides (Grace Bio-labs, Bend, OR) using Aushon 2470 Arrayer (Aushon BioSystems, Billerica, MA) and printed in triplicate (technical replicate). Spotted slides are stored in a sealed Ziploc bag at -20°C. Immunolabeling is performed on an automated slide stainer Autolink 48 (Dako, Carpinteria, CA) according to the manufacturer's instructions (Autostainer catalyzed signal amplification (CSA) kit, Dako, Carpinteria, CA). Each slide is incubated with a single primary antibody at room temperature for 30 min followed by a goat anti-rabbit or mouse IgG secondary antibody. A negative control slide is incubated with antibody diluent instead of primary antibody. The Catalyzed Signal Amplification System kit (Dako Cytomation, Carpinteria, CA, USA) and fluorescent IRDye 680 Streptavidin (LI-COR, Lincoln, Nebraska, USA) are used as the detection system. Total protein for each spot is assessed by staining one in every 20 slides with Sypro Ruby Blot Stain (Molecular Probes, Eugene, OR) according to the manufacturer's directions. Slides are scanned on a GenePix AL4200 scanner (at 635 nm wavelength for antibody slides or 535 nm wave length Sypro Ruby Blot Stained slides), and the images are analyzed by GenePix Pro 7.2 software tools (Molecular Devices, Sunnyvale, CA). The fluorescence signal of each spot is obtained from the fluorescence intensity after subtraction of the local slide background signal.
RPPA Data Normalization Details
Each slide is incubated with a single primary antibody at room temperature for 30 min and followed by either a goat anti-rabbit or anti-mouse IgG secondary antibody depending on the species of the primary antibody. Slides are reacted in batches of 20-30 and each includes one or two negative control slides which are incubated with antibody diluent (negative control slide) instead of primary antibody. Total protein signal for each spot is assessed by staining slides with Sypro Ruby Stain. We select 1 slide in every 20 (in printing order) for total protein staining and use that to perform total protein normalization across all the samples. Normalization method is described below:
A group-based normalization approach is used to normalize the RPPA data. Briefly, for each spot on the array, the-background-subtracted foreground signal intensity (SI) is used as the spot’s SI; if the background intensity is higher than the foreground intensity, the spot’s SI is set to 1 (a very small intensity value). To normalize the antibody SI, the antibody SI of each spot is subtracted by the corresponding SI of negative control and then normalized to the corresponding SI of total protein within the same group. A group of spots are made of the spots that are allocated to the same experiment (or any other condition defined by the investigators). More explicitly, for each spot, the normalized antibody SI can be expressed using the following formula
N = (A – C)*M/T
where N is the normalized antibody SI, A is the antibody SI, C is the negative control SI, M is the median SI of the spots of the same group, and T is the SI of total protein. In addition, for each spot, if the antibody SI is lower than the negative control SI, the normalized SI is set to 1; if the antibody SI, the negative control SI or the total protein SI had a flag indicating the SI is problematic, the normalized antibody SI is set to NA.
Protocol for Antibody Labeling
- Take the slides from freezer and incubate with the following reagents:
- Re-block (15 min)
- I-block (1 hour)
- TBST (0.05M Tris-HCl 0.3M NaCl with 0.1% Tween-20)
- Set up protocol below on the DAKO Autolinker 48 stainer:
- H2O2 (5 min)
- Avidin block (10 min)
- Biotin block (10 min)
- Protein Block (blocking buffer) (5 min)
- Primary antibody (30 min)
- TBST ( (5 min)
- Biotinylated secondary antibody (15 min)
- TBST (5 min)
- SABC (Streptavidin-Biotin Complex)
- TBST (5 min)
- AMP reagent (15 min)
- TBST (5 min)
- Streptavidin-IRDye-680 (LiCor) (15 min)
- dH2O (1 min)
- Take slides out of the stainer
- Rinse twice with dH20 on both sides
- Dry slides
- Scan slides
Slide Spotting Format
a. Experimental lysates
b. Control lysates & other samples:
1) Serial dilution of a known purified protein (0.13 - 32ng/ml) is spiked in 0.5 mg/ml untreated Hela cell lysate
2) Cell lysate mix controls: we have selected four cell lines that express the majority of the antigens to be assayed. The mixed control lysate is serial diluted over a range of 0.0078 mg/ml to 1 mg/ml to test for linear range of antibody response.
- Cell lysates of Pervanadate treated Hela cells mixed with untreated Hela lysate at 0 to 100% for tyrosine phosphorylated antibodies.
- Cell lysates of Calyculin A-treated Jurkat cells mixed with untreated cells at 0-100%. Calyculin A is a potent phosphatase inhibitor which induces threonine phosphorylation.
- NCI 60 cancer cell lines: lysates of selected individual cells and a mix of all/60 cell lines.
- Breast Cancer ATCC 39 cell lines: lysates of selected individual cells and mix of all cells
- Mouse tissue mix: cell lysates from multiple mouse tissues are spotted as positive controls for mouse proteins.
- IgG mix: rabbit, mouse, and goat IgG are mixed at 0.05 mg/ml and spotted at four corners of the slides as gridding and positive controls.
- BSA for non-specific binding controls: one or two different sources of BSA are spotted at 0.5 mg/ml as controls.
- RPPA cell lysis buffer controls.
Total protein by Sypro Ruby staining. The middle slide in every 20 slides printed is stained for total protein with Sypro Ruby and it is used to normalize to total protein. We also stain our first and last slide. For example, in our printing of 240 slides, we stain the following slides: #1, #10, #30, #50.... #230, #240. The antibody labeled slides #2-#19 are normalized by #10 total protein slide while antibody labeled slides #21-#39 are normalized by slide #30 total protein slide, so on and so forth. Negative control slides per run by omitting primary antibody. For each group of 20 slides reacted with different primary antibodies, we react two additional slides without primary antibody but with either anti-mouse or anti-rabbit secondary antibodies and detection Streptavidin-IRDye-680.