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5 min. Blocking Reagent: Bullet Blocking One for Western Blotting

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.

  • Fast blocking in 5 min.
  • Ready to use

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
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
Bullet Blocking One   30 min.
Blocking One 60 min.
Company A
blocking reagent
30 min.
Company B
blocking reagent
60 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 (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.

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
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.

Antibody Diluent 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

Comparison of Blocking Time and Efficiency with 5% Skim Milk

Analysis of Background Noise on Dot Blotting Data

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.

Reference

  1. Nakabo, Shuichiro, et al. "Activated neutrophil carbamylates albumin via the release of myeloperoxidase and reactive oxygen species regardless of NETosis." Modern Rheumatology 30.2 (2020): 345-349.
  2. Kurihara‑Shimomura, Miyako, et al. "Mast cell chymase promotes angiogenesis and lymphangiogenesis mediated by activation of melanoma inhibitory activity gene family members in oral squamous cell carcinoma." International Journal of Oncology 56.5 (2020): 1093-1100.
  3. Ato, Satoru, et al. "Habitual high-protein diet does not influence muscle protein synthesis in response to acute resistance exercise in rat." Nutrition (2020): 110795.
  4. Akiyama, Hiroki, et al. "Control of cell migration by the novel protein phosphatase-2A interacting protein inka2." Cell and Tissue Research (2020): 1-11.
  5. Fujihira, Haruhiko, et al. "Liver-specific deletion of Ngly1 causes abnormal nuclear morphology and lipid metabolism under food stress." Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1866.3 (2020): 165588.

Ordering Information

Product Cat.No. Storage PKG Size Price(US$)  
Bullet Blocking One for Western Blotting 13779-56 4°C 50 ml 25.00 Buy
Bullet Blocking One for Western Blotting 13779-14 4°C 200 ml 40.00 Buy
Bullet Blocking One for Western Blotting 13779-01 4°C 1 L 180.00 Buy

Shipping at room temperature.