新技術：InCellWesternAssay2

III. Experimental Considerations

Proper selection of microplates can significantly affect the results of your analysis, as each plate has its own characteristics including well depth, plate autofluorescence, and well-to-well signal crossover. Use the general considerations for microplate selection provided below.

• In-Cell Western analyses use detection at the well surface with no liquid present. This results in minimal well-to-well signal spread, allowing the use of both clear as well as black-sided plates with clear bottoms. Do not use plates with white wells, since the autofluorescence from the white surface will create significant noise.

• In Cell Western assays require sterile plates for tissue culture growth. The following plates are recommended by LI-COR Biosciences:

96 well format Nunc? (Part Number 161093, 165305)
96 well format Falcon? (Part Number 353075, 353948)

384 well format Nunc? (Part Number 164688, 164730)
384 well format Falcon? (Part Number 353961, 353962)

• The Odyssey Imager requires that microplates have a maximum 4.0 mm distance from the Odyssey scanning surface to the target detection area of the plate. When using the plates specified above for In-Cell Western assays, the recommended focus offset is 3.0 mm.

• If you use plates other than those recommended above, the focus offset can be determined by scanning a plate containing experimental and control samples at 0.5, 1.0, 2.0, 3.0, and 4.0 mm focus offsets. Use the same intensity settings for each scan. After reviewing the collected scans, use the focus offset with the highest signal-to-noise as your focus offset for experiments.

• Protect plates from light before imaging to ensure highest sensitivity. When storing plates after imaging, the plates should remain protected from light at room temperature or 4 °C.

• Intensity for both 700 and 800 nm channels should be set to 5 for initial scanning. If your image signal is saturated or too high, re-scan using a lower intensity setting (i.e., 2.5). If your image signal is too low, re-scan using a higher intensity setting (i.e., 7.5).

• Scan settings of medium to lowest quality, with 169 μm resolution, provide satisfactory results with minimal scan time. Higher scan quality or resolution may be used, but scan time will increase.

• Establish the specificity of your primary antibody by screening lysates through Western blotting and detection on the Odyssey instrument. If significant non-specific banding is present, choose alternative primary antibodies. Non-specific binding of primaries will complicate interpretation of In-Cell Western assay results.

IV. Experimental Results

Figure 1. Dose response of HeLa cells to epidermal growth factor (EGF) as measured by specific antibody detecting dual-phosphorylated ERK (Thr202/Tyr204). The image represents a 96-well two color In-Cell Western with the 800 and 700 channels detecting total and phosphorylated ERK, respectively. Background wells were incubated with secondary antibody but no primary antibody. The graph represents normalized quantitative data demonstrating the percent phosphorylation of ERK.