Blocking Buffers

Product Profile

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.

• Odyssey Blocking Buffer [927-40000, 927-40003, 927-40010, 927-40125, 927-40150]

• Casein Blocking Buffer [927-40200, 927-40300]

• Blocking Buffer Sample Pack [927-40050]

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.

^1. J Immunol Methods 1989, 122 (1), 129-35

^2. Proteomics 2008, 8, 2379–2383 [Figure 1-2]

Optimization

• 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.

^1. J Immunol Methods 1989, 122 (1), 129-35

Technical Resources

Application Notes

• Odyssey Western Blot Blocker Optimization

• One blot Western optimization using the MPX^™ Blotting System

• Good Westerns Gone Bad

• Western Blotting Analysis

Webinars

• “Quantitative, Multiplexed Western Blot Detection with Infrared Fluorescence”
  Amy Geschwender, LI-COR Biosciences

Odyssey Blocker

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

Casein Blocking Buffer

• Ready-to-use formulation in PBS

• Milk-based blockers may contain endogenous:

• Biotin, which could interfere with biotin-streptavidin detection
• Phospho-proteins, which could interfere with phospho-specific antibodies

Blocking Buffer Sample Pack [927-40050]

• Includes:

• 125 ml Odyssey Blocking Buffer (927-40100)
• 100ml Casein Blocking Buffer (927-40300)

• Ideal for empirical testing of blocking conditions

• Identify the blocker that yields the highest signal-to-noise ratio

ELISA Glycoprotein Detection In-Gel Western

Protein Array Quantitative Western Blot Reverse Phase (Lysate) Array

Transcription Factor Assay Western Blot In-Cell™ Western Assay