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After the sample is transferred from the gel to the membrane, the blocking step is performed before the antibody is incubated. Understanding the workings of blocking can improve the quality of western blots, increase data specificity, sensitivity and signal-to-noise ratio.
How does the protein blocking solution work?
The universal PVDF membrane and NC membrane have a strong protein binding capacity, and if the blocking step is skipped, the primary or secondary antibody may bind non-specifically to the membrane, resulting in a high background or "noise point" blot (Figure 1). Blocking buffers "closes" or "closes" these non-specific protein binding sites, ensuring that most of the primary antibodies bind only to the target protein (Figure 2). 
If an effective blocking buffer is chosen, non-specific binding can be reduced without interfering with antibody-antigen binding. 
How to choose the sealing solution?
Choosing the right blocking buffer depends on several factors, including the target of the primary antibody and the detection system being used. Some of the most commonly used blocking buffers contain protein blocking reagents. The protein blocking reagent can be a protein mixture such as skim milk powder or serum, or a single protein such as BSA. Skim milk powder is a popular blocking agent because it is inexpensive and easy to find. However, milk contains phosphoprotein, which may interfere with western blot detection of anti-phosphorylated protein antibodies. Milk can also interfere with biotin-streptavidin-based detection systems. In these cases, a blocking buffer containing BSA may be preferred. Alternatively, to avoid potential cross-reactivity between the antibody and the protein blocking reagent, a protein-free blocking reagent such as PVP-40 can be used.
Azure Fluorescent Blot Blocking Buffer is optimized for use with Azure's versatile imaging system and sapphire dual-mode multispectral laser imaging system to reduce background noise and produce a stable fluorescent signal.
Keep away from noise points: western blots close those things
Why is the Western blot blocking step important?