In previous posts, we’ve talked about Western blot blocking buffers and how important it is to optimize your blocking conditions to get the best results. As many of Western blot users do, you may just routinely use homemade TBS-milk blocking buffer. It’s inexpensive, and it does the job. . . well, most of the time. . .
What you may not know is using milk blocking buffer can cause issues with certain targets. This may give you the wrong information about the presence or the amount of your target. One good way to determine which blocking buffer system to use is to check to see what the primary antibody vendor recommends. Most recommend TBS-based buffer systems. If the primary antibody requires a TBS-based buffer system, we recommend new Odyssey® Blocking Buffer (TBS).
When should you avoid milk blocking buffer?
- When using anti-goat secondary antibodies.
- Reason: Milk contains bovine IgG. Anti-goat secondary antibodies may recognize bovine IgG, resulting in high background.
- When detecting phosphorylated proteins.
- Reason: Milk contains phosphorylated proteins, which may result in low to no signal and high background.
- When using streptavidin-biotin detection systems.
- Reason: Milk contains endogenous levels of biotin. Streptavidin will detect this, resulting in high background.
Here are the results of an experiment evaluating the use of milk and Odyssey Blocking Buffer (TBS). As you can see, milk masked the detection of this protein and is not a good blocking buffer choice.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).
So be sure to optimize your Western blot blocking conditions! The time you spend finding the best blocker will be worth it – and save you from making the wrong conclusions about your experimental data in the future.