Comparison Data: Blocking One (our standard blocking reagent) and 5% skim milk.

Blocking Reagents	Bullet Blocking One	Blocking One	5% Skim Milk
Blocking Time	5 min.	30 min.	60 min.
Blotting Image

Conditions

Comparison Data: Blocking efficiency in 5 minutes

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
	Blocking One		30 min.
	Company A blocking reagent		60 min.
	Company B blocking reagent		30 min.
	Company C blocking reagent （protein-free）		60 min.
	5% Skim Milk in 0.05％ Tween® 20-TBS		60 min.
	3% BSA in 0.05% Tween® 20-TBS		60 min.

Conditions:

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.

Comparison of Blocking Time and Efficiency with 5% Skim Milk

Bullet Blocking One performed better than 5% skim milk in blocking time and efficiency.

Conditions:

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.

Use as Antibody Diluent

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
Blotting Image

Conditions:

Please note;
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.

Comparison with 5% Skim Milk

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.

	Condition 1	Condition 2		Condition 3
Blotting Image
Blocking Reagents	Bullet Blocking One	Bullet Blocking One		5% Skim Milk
Blocking Time	5 min.	5 min.		60 min.
Antibody Diluent	Bullet Blocking One Diluted 20x	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

Detection of a-Tubulin

Chemiluminescence	Fluorescence

Experimental condition

Can Get Signal^® Immunoreaction Enhancer Solution is registered by Toyobo.

Data courtesy from Dr. Eriko Tanaka, Bioscience, Kanagawa Institute of Technology, Japan.

Experimental condition

Can Get Signal^® Immunoreaction Enhancer Solution is registered by Toyobo

Data courtesy from Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan.

Bullet Blocking One for Western Blotting (PDF 63.5 MB)

Bullet Blocking One for Western Blotting (PDF 3.3 MB)