Products

Bullet Blocking One

Bullet Image

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.

Features

  • Fast blocking in 5 min.
  • Ready to use

>> Information on Bullet PAGE One Precast Gel

Product Information

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 Blotting Image Blotting Image Blotting Image

Conditions

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

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

Dot Blotting Data

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.

Applications

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 Blotting Image Blotting Image

Conditions:

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

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 Blotting Image Blotting Image 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

Protocols

Bullet Blocking One for Western Blotting pdf (PDF 62 KB)

Downloads

Bullet Blocking One for Western Blottingpdf (PDF 3.3 MB)

Ordering Information

Product Name Storage Product No. PKG Size Price
Bullet Blocking One for Western Blotting R 13779-14 200 ml e-Nacalai
13779-01 1 L

(Storage) RT: Room temperature, A: Cool and dark, R: Refrigerator, F: Freeze