b'Blocking the membraneThe membrane supports used in western blotting have ablotting use is often system-dependent. Determining the high affinity for proteins. In order to prevent nonspecificproper blocking buffer can help to increase the systems binding of detection antibodies during the steps aftersignal-to-noise ratio. Occasionally, when switching from transfer, it is imperative to block the remaining binding sitesone substrate to another, the blocking buffer that you on the membrane surface.are using will lead to diminished signal or increased background (Figures 18 and 19). A variety of blocking buffers containing milk, normal serum, or highly purified proteins have been used toFor example, with applications using an alkaline block free sites on a membrane. By blocking thesephosphatase conjugate, a blocking buffer in Tris-free sites, the signal-to-noise ratio of the assay shouldbuffered saline (TBS) should be selected because improve through a reduction in background interference.phosphate-buffered saline (PBS) interferes with alkaline Inadequate amounts of blocker will result in excessivephosphatase activity. Likewise, the use of milk as a background noise and a reduced signal-to-noise ratio,blocking reagent should be avoided when using an whereas excessive concentrations of blocker may maskavidinbiotin detection system. Empirically testing various antibodytarget protein interactions or inhibit the markerblocking buffers with your system can help achieve the enzyme, again causing a reduction of the signal-to-noisebest possible results. ratio. The most appropriate blocking buffer for western StartingBlock Blocking Buffer Pierce Clear Milk Blocking Buffer 5% nonfat milk0.3125 g 0.625 g 1.25 gIRAK10.3125 g 0.625 g 0.625 gBcl-xLFigure 18. Comparison of Thermo Scientific StartingBlock Blocking Buffer, Pierce Clear Milk Blocking Buffer, and 5% nonfat milk blocking buffer. Serial dilutions of HeLa lysate were prepared, separated by SDS-PAGE, and transferred to membranes for detection of IRAK1 and Bcl-xL. The membranes were blocked with respective blocking buffers per product instructions. The proteins were probed with Invitrogen primary antibodies against IRAK1 (Cat. No. 4359S) and Bcl-xL (Cat. No. 2764S), followed by an Invitrogen HRP-conjugated goat antirabbit IgG secondary antibody (Cat. No. 32460) diluted per manufacturers instructions. Blots were then incubated with SuperSignal West Atto Ultimate Sensitivity Substrate (Cat. No. A38558). Images were captured at 12 seconds using an Invitrogen iBright imager.Blocker FL 5% milk Blocker FL 5% milk(TBS, 0.05% Tween) (TBS, 0.05% Tween)Wet blots Dry blots(2 days after initial scan)p23 p23Figure 19. Comparison of Thermo Scientific Blocker FL Fluorescent Blocking Buffer and 5% nonfat milk blocking buffer. Western blotting was performed by loading serial dilutions of A431 cell lysate (15 mg, 3-fold serially diluted) and carrying out electrophoresis on an Invitrogen Novex 420% Tris-Glycine Plus Midi Gel (Cat. No. WXP42012BOX). The first 2 lanes were loaded with 5 L PageRuler Prestained Protein Ladder (Cat. No. 26616) and 3 L iBright Prestained Protein Ladder (Cat. No. LC5615). The proteins were transferred to a nitrocellulose membrane using the Power Blotter System (Cat. No. 22834). The membranes were blocked for 30 minutes with either 1X Blocker FL Fluorescent Blocking Buffer (Cat. No. 37565), or TBS with 5% nonfat milk and 0.05% Tween 20 detergent.33'