b'When used with an imager equipped with the appropriateComposite Phospho-ERK1/2 ERK1 GAPDHfilters or lasers, fluorophores with nonoverlapping spectra enable multiplex analysis, in which multiple targets can be detected and independently distinguished in the same blot. This is a unique attribute compared to the popular chemiluminescence-based detection, which would otherwise require multiple rounds of stripping andAlexa Fluor Plus 800Alexa Fluor Plus 680Alexa Fluor Plus 555 reprobing the blot in order to visualize signal from eachsignal signal signalprotein of interest. With each round of stripping andFigure 41. Detection of targets of similar molecular weights. reprobing, one risks stripping protein from the blot, whichFluorescent multiplexing allows for clear distinction of multiple targets would make the comparison between different proteinson the same blot, even when they are of similar molecular weights. A composite image is shown along with images showing the single-of interest challenging. Thus, multiplexing helps makecolor signals of individual proteins. Visualizing the individual signals can research more efficient and productive. For example, onesometimes enable assessment of details that may be harder to see in can visualize a protein of interest simultaneously with aa composite.loading control protein (Figure 40), differentiate proteins ofA431 Cas9similar molecular weights (Figure 41), and evaluate complexEGF A431 control control A431 EGFR KO- + - + - +biological pathways (Figure 42). In multiplexing terminology,160 kDa Compositeprobing for 2 proteins of interest is known as a 2-plex experiment, probing for 3 proteins of interest is known as a160 kDa p-EGFR3-plex experiment, and so on. With the Invitrogen iBright160 kDa EGFRFL1500 Imaging System, one can perform up to a 4-plex 45 kDafluorescent western blot with the appropriate experimental37 kDa Compositesetup (Figure 43).45 kDa p-MEK1Replicate 1 Replicate 2 Replicate 3 45 kDa MEK137 kDa GAPDHCleaved PARP (89 kDa) Figure 42. Evaluation of complex biological signaling pathways. Western blot analysis of the phosphorylation of EGFR and its downstream GAPDH (37 kDa) target, MEK1, after EGF treatment of A431 EGFR knockout (KO) cells.loading control50 gFigure 40. Simultaneous detection of protein of interest and loadingHeLa extract 98 ngcontrol protein. The signal of each protein is captured in a different fluorescence channel, which enables the detection of two proteins on aHA-tagged Rb1 (144 kDa) Alexa Fluor 488single blot without stripping and reprobing. A composite image is shown,HSP90 (86 kDa) Alexa Fluor Plus 800overlaying the signals from each probe.Calreticulin (60 kDa) Alexa Fluor 546p23 (23 kDa) Alexa Fluor Plus 680Figure 43. A 4-plex western blot imaged on an iBright Imaging System. 63'