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 (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
Blotting Image

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.

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

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.

 Bullet Blocking One for Western Blocking (Instruction)

 Bullet Blocking One for Western Blocking (Brochure)

1. Lewis, Mario, et al. "The α-arrestin SUP-13/ARRD-15 promotes isoform turnover of actin-interacting protein 1 in Caenorhabditis elegans striated muscle." PNAS Nexus (2023): pgad330. <<NEW! 

2. Povero, D., et al. "HILPDA promotes NASH-driven HCC development by restraining intracellular fatty acid flux in hypoxia". J Hepatol. (2023). <<NEW!

3. Aoki, Yusuke, et al. "Histone H3K4me3 and H3K9me3 are super over-methylated in soft tissue sarcoma compared to normal muscle in patient-derived xenograft (PDX) mouse models: an indicator of cancer methionine addiction." bioRxiv (2021).

4. Kusama, K., et al. "Endometrial epithelial–mesenchymal transition (EMT) by menstruation-related inflammatory factors during hypoxia." Molecular Human Reproduction 27.6 (2021): gaab036.

5. Obara, Mami, et al. "Expression of cell adhesion molecule, Gicerin/CD146 during the formation of heart and in the cardiac hypertrophy." Molecular and Cellular Biochemistry 476.5 (2021).

6. Kusama, Kazuya, et al. "Cordyceps militaris Fruit Body Extract Decreases Testosterone Catabolism and Testosterone-Stimulated Prostate Hypertrophy." Nutrients 13.1 (2021): 50.

7. Fujita, Kazuyo, et al. "A regulatory role of scavenger receptor class B type 1 in endocytosis and lipid droplet formation induced by liposomes containing phosphatidylethanolamine in HEK293T cells." Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1868.1 (2021): 118859.

8. Pascarelli, Stefano, et al. "Binding of single-mutant epidermal growth factor (EGF) ligands alter the stability of the EGF receptor dimer and promote growth signaling." Journal of Biological Chemistry (2021): 100872.

9. 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.

10. 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.

11. 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.

12. Akiyama, Hiroki, et al. "Control of cell migration by the novel protein phosphatase-2A interacting protein inka2." Cell and Tissue Research (2020): 1-11.

13. 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.