Results obtained from Western blots can be highly variable and dependent not only on the specific antigen/antibody pairs used, but also on other reagents in the protocol such as blocking buffer. Not surprisingly, no one “universal” blocking reagent works well with all proteins. Thus choice of blocking reagent should be assessed for optimal results and having various blockers available in your lab is an advantage. We show here results of Western Blots using Super G blocking buffer.
Super G blocking buffer, although designed for microarray applications, is an effective blocker for Western blot assays on both nitrocellulose and PVDF membranes. Its non-protein based formula was designed for high sensitivity applications on porous membranes. Presented here are results obtained with Western blots after blocking with Super G for the detection of various proteins (GAPDH, PARP, b-Actin, Histone H2A, MAPK). Super G generally produced results comparable to those obtained with a common casein blocker, though in some cases one blocking reagent or another performed better for a specific protein. For example, after blocking with casein Histone H2A barely detected, requiring an extended development time while blocking with Super G produced superior results. In situations such as this when long developing times are required for obtaining visible signal, background levels will also be elevated significantly and may obscure results. In contrast, blots for MAPK required longer development after Super G blocking to obtain comparable results to casein. Further optimization (decreasing blocking time) may remedy this and is on-going further trials with Super G, but speaks to the variability expected from antigen to antigen with different blockers.
Super G Blocking Buffer is compatible with direct detection methods such as fluorescence as well as enzyme-linked detection methods using alkaline phosphatase (as shown here) and horseradish peroxidase (data not shown). It is very efficient at blocking porous membranes and, in most cases, dilutions up to 10-fold in 1x PBS were used for adequate blocking. Additionally, shorter blocking times are possible down to 10 min. (data not shown). In general, blocking with Super G produced excellent results for Western blots and may facilitate detection for some proteins over other blockers.
Figure 1. Detection by Fluorescence and Colorimetric Methods on PVDF Membranes
Figure 1. Typical results achieved with Super G blocking reagent compared to casein blocking on Western blots using fluorescent and colorimetric endpoint detection on PVDF membranes. For all panels, 500 ng of mouse liver lysates were separated by SDS-PAGE and transferred to Millipore Immobilon-FL (fluorescent) and Immobilon-P (colorimetric) PVDF membranes. Membranes were blocked for 1 hour with respective blockers (Panel A: 0.1x Super G in 1x PBS; B: Thermo Casein Blocker; C: 0.5x Super G; D: 0.25x Super G; E: 0.1x Super G). Blocking was followed by detection with a monoclonal anti-GAPDH antibody (1:1000) overnight in 1x PBST followed with (A) 1:10,000 anti-goat IgG-Alexa-647 and –Alexa555 or (B-E) colorimetric endpoint detection utilizing alkaline phosphatase with BCIP/NBT substrate (VECTASTAIN ABC System, Vector Laboratories) after a 5 minute development time. A band at 37 kDa is detectable on all blots.
Figure 2. Detection by Colorimetric Methods on Nitrocellulose Membranes
Figure 2. Typical results achieved with Super G blocking reagent compared to casein blocking on Western blots using colorimetric endpoint detection on nitrocellulose membranes. For all panels, 500 ng of mouse liver lysates were separated by SDS-PAGE and transferred to Whatman Protran nitrocellulose membranes. Membranes were blocked for 1 hour with respective blockers (0.1x Super G in 1x PBS or Thermo Casein Blocker) followed by detection with various antibodies (1:1000) overnight in 1x PBST/0.25% BSA (Panel A: monoclonal anti-p44/42 MAPK; Panel B: polyclonal anti-b-Actin; Panel C: monoclonal anti-PARP; Panel D: polyclonal anti-Histone H2A). Visualization was performed with colorimetric endpoint detection utilizing alkaline phosphatase with BCIP/NBT substrate (VECTASTAIN ABC System). Development times varied for antibody and blocker as follows: Panel A – 60 min. (Super G) and 8 min. (casein); Panel B – 10 min. (Super G) and 3 min. (casein); Panel C – 15 min. (Super G) and 5 min. (casein); and Panel D – 15 min. (Super G) and 93 min. (casein). Expected bands were visible for all blots: (A) 42 and 44 kDa MAPK, (B) 45 kDa b- Actin, (C) 116 kDa PARP, and (D) 12 kDa Histone H2A.