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Optimizing Your Blocking Buffer

Effect of various blocking agents on detection of pAkt and total Akt in Jurkat lysate

Figure 1. Effect of various blocking agents on detection of pAkt and total Akt in Jurkat lysate after stimulation by calyculin A. Total and phosphorylated Akt were detected in calyculin A-stimulated (+) and non-stimulated (-) Jurkat lysate at 10 µg; 5 µg; and 2.5 µg/well. Blots were probed with pAkt Rabbit mAb (Santa Cruz P/N sc-135650) and Akt mAb (CST P/N 2967) and detected with IRDye® 800CW Goat anti-Rabbit IgG (LI-COR P/N 926-32211) and IRDye 680RD Goat anti-Mouse IgG (LI-COR P/N 926-68070); scanned on Odyssey® CLx (auto scan 700 & 800). pAkt (green) is only detected with Odyssey Blocking Buffer (TBS).

Determining the best blocking agent for each target will provide the best chance for success. Below are protocols to help you optimize your Western blot conditions in one experiment.

Technical Resources

Application Notes

Quick Tips To Help You Get Started

  • Check to see what the primary antibody vendor recommends. Most primary antibody vendors recommend TBS-based buffer systems. If the primary antibody requires a TBS-based buffer system, we recommend Odyssey Blocking Buffer (TBS).
  • Some blocking buffers may interfere with detection. Milk protein blockers, for instance, may contain endogenous biotin or phosphorylated proteins, which result in high background.
  • Compare signal intensity (positive control) with membrane background or negative control.
  • If target signal intensity is low, blocking could be too efficient. Try a different blocking buffer.
  • Overblocking with milk (at high concentrations or for long periods) can cause general loss of blotted proteins from the membrane1.


  1. J Immunol Methods 1989, 122 (1), 129-35
  2. Proteomics 2008, 8, 2379–2383 [Figure 1]

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