b'SuperSignal West Atto substrate allows use of very dilute samples. p23 in whole-cell HeLa cell lysates was detected with SuperSignal West Atto, Bio-Rad Clarity, or Millipore Immobilon substrate. Only 1.25 g of cell lysate was needed to get a strong signal with SuperSignal West Atto substrate, whereas 20 g of cell lysate was required to get the same level of detection with the other two substrates (Figure 31).A B20 10 5 2.5 1.25 0.625 0.312 0.156 0.078 0.04 g 1,000900800Relative band intensity(background-corrected)700SuperSignal West Atto 600500Bio-Rad Clarity 400300Millipore Immobilon 2001000SuperSignal West Atto Bio-Rad Clarity Immobilon Western1.250.04 g vs. 200.625 g 200.625 gFigure 31. SuperSignal West Atto substrate allows use of very dilute samples. HeLa cell lysates were separated on Novex Tris-glycine gels and transferred to nitrocellulose membranes using the Power Blotter System with Power Blotter Select Transfer Stacks. The blots were incubated with mouse anti-p23, followed by an HRP-conjugated goat antimouse IgG secondary antibody. Finally, respective blots were incubated with SuperSignal West Atto substrate, Clarity Western ECL Substrate (Bio-Rad), or Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore) per product instructions. All blots were simultaneously acquired using the iBright FL Imaging System.Did you knowA speckled background on film can be caused by aggregate formation in the HRP conjugate. Filtering the conjugate through a 0.2 m filter may help, or use a new HRP conjugate.51'