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. 2014 Sep 16;86(20):10429–10436. doi: 10.1021/ac502932t

Figure 3.

Figure 3

In situ chemical lysis of cells in microwells at elevated temperatures and with enclosed microwells notably improves scWestern performance. (A) False-color fluorescence micrographs and intensity profiles show scWesterns after cool (left, 4 °C) and hot (right, 50 °C) lysis buffer conditions, suggesting a fully resolved protein pair, GAPDH (magenta signal) and β-tubulin (blue signal) under the 50 °C lysis conditions. (B) Fluorescence micrographs during in-microwell lysis of U373-GFP cells under 4 and 50 °C lysis conditions in systems with (enclosed) and without (open) a lid covering the microwell, suggest an enclosed microwell architecture can mitigate protein losses during cell lysis. (C) Time course of the total integrated GFP fluorescence signal from each microwell in part B. Error bars from 3 to 5 independent experiments indicate standard deviation. E = 40 V cm–1, lysis time = 20 s, electrophoresis time = 40 s, 10%T PA gel. (GAPDH, Alexa Fluor 555-labeled secondary antibody; β-tubulin, Alexa Fluor 647-labeled secondary antibody).