Basal profile of receptor tyrosine kinase signaling network measured by ELISA - Dataset (ID:20137)

HMS Dataset ID: 20137
Dataset Title: Basal profile of receptor tyrosine kinase signaling network measured by ELISA
Publication(s) Using Dataset: PMID: 24065145; PMID: 24655548
Project Summary Page(s):;
Screening Lab Investigator: Mario Niepel
Screening Principal Investigator: Peter Sorger
Assay Description: Here, basal profiles for cells are the data for the abundance and basal phosphorylation of 22 receptors and 3 downstream kinases, as well as for the abundance of estrogen receptor (ER) and progesterone receptor (PR). In this dataset, basal profiles were obtained for approximately 40 breast cancer cell lines. All basal phosphorylation and protein abundance data were obtained by ELISA and, where possible, we estimated steady-state levels of molecules per cell using calibrated, recombinant standards. For the receptor tyrosine kinase phosphorylation data, we measured the amount of total phosphotyrosine rather than of individual phosphorylation sites, with the exception of IGF1R, for which we measured phosphorylation of Tyr1131.
Assay Protocol: 1. Cells were plated either in nine 15-cm dishes and grown as described in Niepel, Hafner et al. 2013 (PMID: 24065145).
2. Cells were grown for 24 hours and then serum starved in base media without additives for an additional 18 hours.
3. Cell numbers were chosen to yield approximately 75% confluency at the time of lysis.
4. Plates were washed with 12 ml of ice cold PBS and drained for 30 sec.
5. Two plates were treated for approximately 5 min with 1.5 ml 0.05% Trypsin (Corning, 25-052-CI), until cells detached from the plates.
6. Tryspin was stopped with 8.5 ml of 10% FBS in PBS and gently pipetted up and down until they were in single-cell suspension.
7. Cells from each plate were counted in duplicate using a Cellometer Auto T4 (Nexcelcom).
8. Seven plates were lysed with 1 ml of lysis buffer containing: Mammalian Protein Extraction Buffer (M-PER; Thermo Scientific, 78501) supplemented with protease inhibitor cocktail (Sigma-Aldrich, P2714), 1 mM sodium orthovanadate (Sigma-Aldrich, S6508), 5 mM sodium pyrophosphate (Sigma-Aldrich, 221368), 50 µM oxophenylarsine (EMD Biosciences, 521000), and 10 µM bpV (phen; EMD Biosciences, 203695).
9. Lysed cells were scraped off the plate, collected in microcentrifuge tubes, and incubated on ice for 30 min.
10. Membranes and cell debris were sedimented by centrifugation at 20,000g for 10min at 4°C, the supernatants were pooled, aliquoted, and subsequently stored at -80°C.
11. For protein measurements, 384 well, black, flat-bottomed, polystyrene, high-binding ELISA plates (Corning, 3577) were incubated overnight at room temperature with capture antibodies. Antibodies are described in Niepel, Hafner et al. 2013 (PMID: 24065145).
12. Plates were then blocked with 2% bovine serum albumin (BSA) in PBS for 1 hour.
13. Plates were washed four times with 0.05% Tween-20 in PBS (PBS-T) then incubated with lysates and recombinant protein standards for 2 hours at room temperature.
14. After each antibody incubation the plates were washed four times with PBS-T.
15. ELISAs were incubated with primary and secondary antibodies for 2 hours and 1 hour, respectively. Antibodies are described in Niepel, Hafner et al. 2013 (PMID: 24065145).
16. All ELISAs were visualized with SuperSignal ELISA Pico Chemiluminescent Substrate (Pierce, 37069) and luminescent signal was measured with an EnVision Plate Reader (Perkin Elmer).
17. Data from ELISA measurements was extracted using MATLAB Statistical Toolbox.
18. Raw data were background subtracted by subtracting raw values of measuring assay buffer from raw values of each sample measurement.
19. A dilution series of recombinant protein (standard curve) was used to convert raw signal into known protein concentration (pg/ml).
20. A linear best fit line was calculated based on the standard curve, then background subtracted values for each sample were converted into pg/ml using the linear equation (y = mx + b) of the standard curve.
21. Regressed data were multiplied by the dilution factor for each sample, then mean and standard error of the mean values were calculated for at least four technical replicates for the basal profile measurements and two biological replicates for the ligand response measurements.
22. Data above or below the range of detection was set to the upper or lower detection limit, respectively.
23. For steady-state protein levels, ELISA measurements (pg/ml) were normalized to the number of cells in the lysate (cells/ml) to get pg protein per cell.
Assay Protocol Reference: Niepel M, Hafner M, Pace EA, Chung M, Chai DH, Zhou L, Schoeberl B, Sorger PK. Profiles of basal and stimulated receptor signaling networks predict drug response in breast cancer lines..Sci Signal. 2013 Sep 24;6(294):ra84. doi: 10.1126/scisignal.2004379. PMID: 24065145
HMS Dataset Type: ELISA
Date Publicly Available: 2013-09-24
Most Recent Update: 2016-11-08