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.
|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.
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).
Kusama, K., et al. "Endometrial epithelial–mesenchymal transition (EMT) by menstruation-related inflammatory factors during hypoxia." Molecular Human Reproduction 27.6 (2021): gaab036.
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).
Kusama, Kazuya, et al. "Cordyceps militaris Fruit Body Extract Decreases Testosterone Catabolism and Testosterone-Stimulated Prostate Hypertrophy." Nutrients 13.1 (2021): 50.
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.
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.
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.
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.
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.
Akiyama, Hiroki, et al. "Control of cell migration by the novel protein phosphatase-2A interacting protein inka2." Cell and Tissue Research (2020): 1-11.
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.
Shipping at room temperature.