Table 2.

Comparison of Traditional Western Blot and Differential Sensing Array

time (h)	differential sensing array	time (h)	Western blot analysis36,37
0.5	Solutions preparation: (l) 2× assay buffer (50 mM HEPES buffer (pH = 7.4), 100 mM KC1, 0.2 mM EDTA, 0.2 mM EGTA, 4 mM DTT and 20 mM MgCl2); (2) cell lysate; (3) 2× substrate solution (10 μM of each sensor and 1 mM ATP)	2.5	(l) mix 60—70 μg of each lysate or different amounts of recombinant activated target kinase with 5× SDS-PAGE loading dye and 1× lysis buffer, boil for 5 min then load into the wells of the gel; (2) gel electrophoresis at 120 V for 1—1.5 h
1	plate preparation: (l) training set preparation, addition of increasing concentrations of recombinant activated target kinase and the cell lysate (7 μg/well) in the assay buffer; (2) test set preparation, cell lysates (7 μg/well) diluted in the assay buffer and loaded into the same plate to quantify the target kinase	2.5–15.5	transfer at 90 V for 2 h or at 30 V for overnight at 4 °C then wash 3 × 5 min
1.5	readout: (l) plate reader injector is used to add the substrates solution into the wells that contain the lysates diluted in the assay buffer. (2) recording of changes in the emission (ex.360 nm; em.485 nm) over time	2.5–16.5	membrane block for 1 h at room temp then incubation with primary Ab for 1 h at room temp or overnight at 4 °C; wash 5 × 5 min
0.5	data analysis: (l) curve fitting (calculation of rate constants); (2) chemometrics (LDA); (3) developing a calibration model or training set; (4) prediction of unknown kinase concentrations in the test set	1.5	incubation with secondary Abs for 1 h at room temp, wash 5 × 5 min
		1	ECL reagent treatment and film development or Licor System analysis
		1	data analysis
~3.5	total	11–38	total