The identification of compounds that alter protein levels has required cumbersome in-vitro analyses. This procedure involves treating cultured cells with individual drugs, followed by detergent lysis, polyacrylamide gel electrophoresis of total cellular proteins, and Western blotting to determine protein levels. This methodology is decidedly unsuitable for rapid compound analysis. In contrast, the Her2-blot, which relies on whole-cell immunodetection of the desired proteins, utilizes a minimal number of cells, yet it is sufficiently sensitive and reproducible to permit quantitative determinations. The assay is a hybrid of a Western blot and an enzyme-linked immunosorbent assay (ELISA), and is a modified version of the cytoblot assay developed by Stockwell et al. (12
). The cytoblot assay is adaptable for screening numerous protein modifications because the only requirement is a specific primary antibody directed against the protein of interest. It has been applied to HTS to identify many bioactive small molecules, such as inhibitors of mitotic spindle bipolarity (13
), mitosis modulators (14
), inhibitors of phospho-STAT3 (15
), inhibitors of HDAC6-mediated tubulin deacetylation (16
), and modulators of the human chromatid decatenation checkpoint (17
). Our contribution to the cytoblot is that for the first time we detected a specific decrease in the amount of a protein (9
). Testing for the presence of a gene product is far simpler than testing for the absence or near absence of a gene product, because the amount of protein presumably increases with time in the first instance, and thus distinguishes itself from the background. In contrast, when the disappearance of a product is measured, the best resolution from background is at time zero. Notwithstanding this theoretical difficulty, meaningful measurements of Her2 can be obtained.
shows a schematic representation of the assay. The method consists of plating cells in microtiter dishes and treating wells with small molecules at equal concentrations, to identify agents that alter cellular levels of the protein, or at a concentration range to determine their potency in degrading Her2. Following treatment, cells are fixed and permeabilized with methanol. An antibody against the protein and a secondary antibody linked to horseradish peroxidase (HRP) are added. Upon addition of a luminescent substrate, the signal emitted is read in a luminometer. In an assay such as this, where a reduction in signal represents a hit, there are potential sources for false positives, such as from cytotoxic agents, which induce a decrease in the signal by cell death rather than through Her2 modulation. Thus, during screening it is important to identify cytotoxic compounds in the library that may reduce total cell number, rather than having a specific effect on Her2. For this reason, the assay can be multiplexed with an indicator of cell viability. A low-cost alternative to viability assays is to determine the amount of total protein in each well using the bicinchoninic acid (BCA) assay.
The Her2-assay described later is designed to include the following controls: vehicle-treated wells (control), wells containing cells treated with geldanamycin or radicicol (positive control; the signal should reach background levels), and wells containing only detection reagent (set as blank in SoftMaxPro software; the values will be subtracted from all analyzed wells). To control for nonspecific antibody binding to the plate, a normal rabbit IgG is added to the plate (background).
- Dispense the compound library or vehicle (DMSO) to wells, and incubate plates for 12 h (or 6 h) at 37°C and 5% CO2 (see Note 11). Use two wells for the positive control. For geldanamycin and radicicol, the final concentration of the compound in the well is 50 nM.
- Remove media through aspiration and wash each well with ice-cold TBST (2 × 200 μL). Use a vacuum source (such as house vacuum) attached to an eight-channel aspirator to remove the liquid from plates.
- Add cold methanol (100 μL at -20°C) to each well, and incubate the plate at 4°C for 10 min.
- Wash each well with TBST (2 × 200 μL) to remove the methanol, and incubate at RT for 2 h with 200-μL Super-Block.
- Add the anti-Her-2 (c-erbB-2) antibody (100 μL; 1:200 in SuperBlock) to each assay well and a normal rabbit IgG (100 μL; 1:200 in SuperBlock) to nonspecific-binding control wells and incubate the plate overnight at 4°C with gentle rocking.
- Wash each well with TBST (2 × 200 μL), add the anti-rabbit HRP-linked antibody (100 μL; 1:2,000 in SuperBlock), and incubate the plate at RT for 2 h.
- Wash with TBST (3 × 200 μL) to remove excess antibody (see Note 12).
- Add ECL™ Western Blotting Detection Reagents 1 and 2 in a 1:1 mixture (100 μL) (see Note 13). Read the plate immediately in an Analyst GT plate reader. Scan each well for 0.1 s. Luminescence height = 0.6 mm; maximum integration = 100,000 μs.
- Settings: readings per well = 1; target CV per well = 1.0%; raw data units = counts/sec; attenuation mode = out; motion settling time = 15 ms; luminescence aperture = 384/96 aperture; max cps = 100,046.9922 cps; min counts = 349 counts.
- Import and analyze data in SoftMaxPro (Molecular Devices). Subtract readings from IgG control wells from all measured values. Calculate % Her2 level as luminescence readings resulting from drug-treated cells/untreated cells (vehicle treated) × 100 (9). shows representative raw data obtained from a Her2-blot (see Note 14).
Fig. 10.3 Representative results of a Her2 blot. Readings obtained in wells containing SKBr3 cells treated for 12 h with DMSO only (control) (average 141715.3; s.d. 8573.8), 50 nM radicicol (RD) (average 9815.3; s.d. 2459.1), and 50 nM geldanamycin (GM) (average (more ...)
- To determine the protein content in each well, wash plates from Her2-blot readings with TBST (2 × 200 μL) and incubate them with the BCA reagent (150 μL) for 30 min at 37°C. Add a standard solution of BSA to the blank wells.
- Read absorbance using the Analyst GT (Molecular Devices). Instrument settings: read sequence = row; mode sequence = well; detection mode = a9; excitation side = top; emission side = top; lamp = continuous; top dichroic = 50:50 beam splitter; readings per well = 1; integration time = 100,000 μs; motion settling time = 200 ms; Z height = 5.33 mm, middle; excitation filter = fluorescence 530 nm. Import and analyze data in SoftMaxPro (Molecular Devices) and determine the protein content of each well (see Note 15).