Western QuickBlock Kits

In immunological assays such as Western blot and ELISA, widely used fat-free milk is effective in blocking for up to 80% of antibodies in a routine one-hour procedure. The remaining 20% of antibodies, however, cannot be appropriately blocked with fat-free milk due to cross reactivity. GenScript developed Western QuickBlock Kit (L00276) for five-minute speedy blocking of most antibodies as effective as fat-free milk, and Western QuickBlock Optimization Kit (L00278) for quick selection of best blocking reagent for the antibodies that fat-free milk is ineffective in blocking. GenScript's Western QuickBlock Kit is designed for the reliable and speedy blocking of any membranes used for dot and Western blots. It can also be used to block any other solid surface, such as the microtiter plates used for ELISA. Instead of the classical one-hour blocking procedure, this kit enables the complete blocking of the solid surface in just five minutes. Furthermore, the QuickBlock method has been shown to increase the sensitivity of Western detections when compared to the dry milk method, allowing the researcher to reach the same antigen detection level using smaller amounts of antibody.

Figure 1. Western blots for the detection of GAPDH protein from Hela cell lysate by using either the classical 10% dry milk blocking or the Western QuickBlock Kit. 5.0 μg, 1.7 μg, and 0.6 μg of Hela cell lysate (BD Biosciences, #611449) were loaded in different lanes as shown in the figure. Both of the Western blots were developed with LumiSensorTM Chemiluminescent HRP Substrate (GenScript, L00221V60).

In some cases, the sequence similarity between an antigen and the blocking reagents causes high background in Western blots, Dot blots, and ELISA, sometimes so much that the antigen cannot be detected clearly. To solve this problem, GenScript has introduced the Western QuickBlock Optimization Kit. With four different QuickBlock reagents provided in each kit, researchers can quickly find the best reagent combination for any primary antibody. GenScript provides Customized Western QuickBlock Optimization Kit that each contains only one of the four blocking reagents for already optimized blocking.

Key Features:

• Quick procedure: The QuickBlock system takes only five minutes.
• Increased sensitivity: QuickBlock makes your Western detection more sensitive.
• Quick optimization: Finding the best blocking reagents now takes a few hours instead of a few days.
• Ease of performance: We offer a quick and simple procedure.

Storage:

Store the kit at 4°C. It will remain stable for six months.

Examples:

An act of blocking using the Western QuickBlock Kit is here compared to the classical 10% dry milk blocking technique in a Western blot detection of GAPDH protein from Hela cell lysate. Serially diluted Hela cell lysate samples were Western-blotted to WestClear^TM nitrocellulose membrane after SDS-PAGE. The membrane was then cut into two halves and processed with the same procedures using polyclonal goat anti GAPDH antibody (GenScript, A00191). The choice of blocking reagents was the only between the two: Classical 10% dry milk blocking (one hour, left panel of Figure 1), and Western QuickBlock Kit (five minutes, right panel of Figure 1).

Figure 1. Western blots for the detection of GAPDH protein from Hela cell lysate by using either the classical 10% dry milk blocking or the Western QuickBlock Kit. 5.0 μg, 1.7 μg, and 0.6 μg of Hela cell lysate (BD Biosciences, #611449) were loaded in different lanes as shown in the figure. Both of the Western blots were developed with LumiSensorTM Chemiluminescent HRP Substrate (GenScript, L00221V60).

The Western QuickBlock Optimization Kit was used to optimize the western blot detection of α-Tubulin in Hela cell lysate. 10 μg of Hela cell lysate was electrophoresised in four different lanes of SDS-PAGE and western-blotted onto WestClear^TM nitrocellulose membrane. The membrane was then cut into four longitudinal strips and processed using the same procedures using unpurified monoclonal anti-α-Tubulin (Mouse Ascites Fluid, Sigma, T5168). The choice of blocking reagents is the only difference between the two.

Figure 2. Western blots for the detection of α-Tubulin in Hela cell lysate by using the Western QuickBlock Optimization Kit. 10.0 μg of Hela cell lysate (BD Biosciences, #611449) was loaded in each of the four different lanes as shown in the figure. Monoclonal anti-α-Tubulin (Mouse Ascites Fluid, Sigma, T5168) was used with the Western Optimization Kit (L00258), which contains all the components of the Western QuickBlock Optimization Kit.

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