LI-COR offers several blocking buffers for use with the Odyssey® and Aerius Systems. All are optimized for use with IRDye® products and other near-infrared fluorophores and provide excellent performance for quantitative Western blots and other immunoassays.
Blocker choice is important for immunoassay success. Blocking buffers enhance sensitivity by reducing background interference, increasing signal-to-noise ratio, and promoting specific binding of the primary antibody while minimizing non-specific interactions.
Insufficient blocking yields high background and reduced signal-to-noise ratios.
Excessive blocking may cause loss of blotted proteins1 or mask antibody:antigen interactions.
Detection reagents may cross-react with certain blocking buffers.
Empirical testing is critical! The Blocking Buffer Sample Pack provides smaller quantities of two LI-COR blockers, to help you identify the best choice for your antigen.
No single blocking buffer selection is suitable for ALL antigen-antibody pairs. Blocker choice may affect antibody specificity and non-specific binding2.
[LEFT] PKC-a Western blots of tissue lysates, illustrating significant differences in antibody performance between 5% BSA blocker and Odyssey Blocking Buffer .
[LEFT] pAkt and ß-Tubulin antibody specificity depends on blocker selection. In 5% BSA, the pAkt antibody shows extra bands (green); in I-Block™ (Applied Biosystems), ß-Tubulin shows extra bands. In Odyssey Blocker, antibody specificity is increased.
Try several blocking buffers to see which produces the best signal-to-noise ratio.
- We recommend that you start with Odyssey Blocking Buffer. If necessary, other blocking buffers can then be tested.
- In our hands, BSA blockers often cause increased non-specific binding.
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.
Some blocking buffers may interfere with detection. Milk protein blockers, for instance, may contain endogenous biotin or phospho-proteins.
Overblocking with milk (at high concentrations or for long periods) can cause general loss of blotted proteins from the membrane1.
Odyssey Blocking Buffer: the gold standard for quantitative Western blot detection
Contains no mammalian proteins, for the lowest cross-reactivity with mammalian antibodies
Ready-to-use formulation in PBS
Excellent choice for biotin-streptavidin detection
Suitable for a wide range of applications, including Western blots, In-Cell Western™ assays, and protein arrays
[LEFT] Detection of phospho-ERK in serial dilutions of A431 lysate (5 µg to 78 ng). Unstimulated lysates are shown to the left of the MW marker; EGF-stimulated lysates are on the right. Odyssey Blocking Buffer was used for blocking and antibody dilution.
Primary Ab: rabbit anti-phospho-ERK (CST 9101); secondary Ab: IRDye 680 goat-anti-rabbit (LI-COR 926-32221).
[LEFT] Detection of p53 in serial dilutions of A431 lysate (5 µg to 78 ng). Casein was used for blocking and antibody dilution. Primary Ab: mouse anti-p53 (Sigma P6874); secondary Ab: IRDye 800CW goat-anti-mouse (LI-COR 926-32210).