Ready-to-use / 5 min.
Bullet Blocking One is a blocking reagent for western blotting, which exhibits excellent blocking efficiency in only 5 minutes due to the amphiphilic compound used.
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|Blocking Reagents||Bullet Blocking One||Blocking One||5% Skim Milk|
|Blocking Time||5 min.||30 min.||60 min.|
|Sample:||20 µg of HeLa cell extract, 5 serial two-fold dilution series|
|SDS-PAGE:||Bullet PAGE One 5-15% (Product No. 13080) with SDS Running Buffer (Product No. 30329) at 400 V for 10 min.|
|Blotting:||Semi-dry Blotting Buffer Solution (Product No. 30650) at 10 V for 30 min|
|Washing:||0.1% t-TBS (Product No. 12750)|
|Blocking:||Refer to the figure above|
|1st antibody:||Anti-Vimentin (C-20) (Rabbit) (Product No. SC-7557-R) diluted 1:2,000, 1 hr. at RT|
|2nd antibody:||Anti-Rabbit IgG-HRP (Product No. SC-2004) diluted 1:100,000, 1 hr. at RT|
|Detection:||Chemi-Lumi One (Product No. 11644), 5 min. reaction time|
|Detector:||LAS-3000 (High mode), 15 min. exposure time|
The original Blocking One, the competitors' ready-to-use blocking reagents and the conventional blocking reagents did not show enough blocking efficiency in 5 min, while Bullet Blocking One performed well.
|5 min. Blocking Time||Blocking Reagents||Manufacturers' Recommended Blocking Time|
|Bullet Blocking One||30 min.|
|Blocking One||60 min.|
blocking reagent （protein-free)
|5% Skim Milk
in 0.05% Tween® 20-TBS
in 0.05% Tween® 20-TBS
PVDF membranes dot-blotted with mouse serum were washed with TBS. Blocking was performed using each reagent above. Anti-mouse IgG (Cat.#SC-2005) (1:5,000 in 0.01% t-TBS) was applied, and the membranes were washed with 0.05% t-TBS. After reaction with Chemi-Lumi One Super (Cat.#02230), detection was performed using LAS-3000 (High mode) with 90 sec. exposure time.
Diluting antibody with Bullet Blocking One resulted in less non-specific binding compared to using t-TBS as diluent.
|Antibody Diluent||Bullet Blocking One (Undiluted)||0.1% t-TBS|
|Sample:||10 µg of HeLa cell extraction, 3 serial two-fold dilution series|
|SDS-PAGE:||Bullet PAGE One 5-15% (Product No. 13080) with SDS Running Buffer (Product No. 30329) at 400 V for 12 min.|
|Blotting:||Semi-dry Blotting Buffer Solution (Product No. 30650) at 10 V for 30 min.|
|Washing:||0.1% t-TBS (Product No. 12750)|
|Blocking:||Bullet Blocking One, 5 min.|
|1st antibody:||Anti-Cox4 (D-20) (Goat) (Product No. SC-69359) diluted 1:500, 1 hr. at RT|
|2nd antibody:||Anti-Goat IgG-HRP (Product No. SC-2350) diluted 1:5,000, 1 hr. at RT|
|Detection:||Chemi-Lumi One Super (Product No. 02230), 1 min. reaction time|
|Detector:||LAS-3000 (High mode), 10 min. exposure time|
For antibody dilution, use Bullet Blocking One undiluted, or dilute up to 20x with TBS or PBS containing 0.05 – 0.1% detergent, such as Tween 20. The appropriate dilution ratio depends on antibody conditions, such as type and concentration, pretest is required.
Bullet Blocking One showed good blocking efficiency in 5 min. Although optimizations of dilution ratio to types of antibody are required beforehand, in this experiment, diluting the antibody with Bullet Blocking One resulted in the strongest signal.
|Antibody Diluent||Condition 1||Condition 2||Condition 3|
|Blocking Reagents||Bullet Blocking One||Bullet Blocking One||5% Skim Milk|
|Blocking Time||5 min.||5 min.||60 min.|
|Antibody Diluent||Bullet Blocking One
|5% Skim Milk||5% Skim Milk|
|1st Antibody||Anti-UCP1 Diluted 2,000x Room temperature, 1 hr.|
|2nd Antibody||Anti-Rabbit IgG Diluted 2,000x Room temperature, 1 hr.|
Data courtesy of Associate Professor Goto Tsuyoshi / Kim Minji, Functions of Food that Support Everyone's Health, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University
Dot Blotting Data
PVDF membranes dot-blotted with mouse serum were washed with TBS. Blocking was performed using each reagent above. Anti-mouse IgG (Product No. SC-2005) (1:5,000 in 0.01% t-TBS) was applied, and the membranes were washed with 0.05% t-TBS. After reaction with Chemi-Lumi One Super (Product No. 02230), detection was performed using LAS-3000 (High mode) with 90 sec. exposure time. Data analysis was done with Multi Gauge.
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Shipping at room temperature.