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).