30 min Antigen-Antibody Reaction: Bullet ImmunoReaction Buffer
When used for immunoblotting, Bullet ImmunoReaction Buffer may shorten antigen-antibody reaction time by half.
- Saving-time: Cut in half antigen-antibody reaction time
- Simple: Replace tTBS, tPBS, skim milk or BSA with Bullet ImmunoReaction Buffer
- Ready to use: Do not need to dilute
Application 1
Gly m4, a major soybean allergen
| Saving-time protocol | Conventional protocol | |||
| Western blotting |  |
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| Antibody diluent | Bullet ImmunoReaction Buffer | 5% skim milk | Bullet ImmunoReaction Buffer | 5% skim milk |
| Primary antibody:Anti-Gly m4 rabbit in-house polyclonal antibody(ST0225), X 1,000 dilution | 30 minutes (Room temperature) | Overnight at 4°C | ||
| Secondary antibody: Stabilized Peroxidase conjugated Goat anti-rabbit(H+L) manufactured by Thermo Fisher, X 400 dilution | 30 minutes(Room temperature) | 60 minutes (Room temperature) | ||
Experimental details
| Sample | Commercially available soy milk |
| Blocking | Incubated for 60 minutes with 5% skim milk |
| Detection | ECL manufactured by GE on X-ray film |
| Exposure Time | 8 minutes for saving-time protocol / 1 minute for conventional protocol |
Data courtesy of Professor Tatsuya Moriyama and Dr. Erika Yano, Faculty of Agriculture, Department of Applied Biological Chemistry, Kinki University
Reference Data
Comparison of sensitivity related to Ab-Ag reaction time with 0.1% tTBS
| Primary Ab incubation time | 15 min | 30min | 60 min | 120 min | Overnight at 4C |
| Secondary Ab incubation time | 15 min | 30min | 60 min | 120 min | 60 min |
| Bullet ImmunoReaction Buffer |  |
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| 0.1%-tTBS |  |
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Experimental details
| Sample | Hela cell extract; (left) 2 μg, (right) 0.4 μg |
| Blocking | Incubated for 60 minutes with 5% skim milk |
| Primary antibody | β-Actin antibody manufactured by Santa Cruz, #sc-47778, X 1,000 dilution |
| Secondary antibody | Goat anti-mouse IgG(H+L) secondary antibody manufactured by Novous, NB7574, X 40,000 dilution |
| Detection | Chemi-Lumi One Super manufactured by Nacalai, #02230, 5-minute exposure time |
| Detector | LAS-300 High mode |
Application 2
Comparison of detection of β-Actin protein with 0.1% tTBS and 5% skim milk
| Antibody diluent | Bullet ImmunoReaction Buffer | 0.1%-tTBS | 5% skim milk |
| Western blotting |  |
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| Primary antibody:Anti-β-Actin rabbit manufactured by CST, X 5,000 dilution | 20 minutes | 60 minutes | |
| Secondary antibody:anti-rabbit IgG(H+L) secondar antibody manufactured by Novous, X 5,000 dilution | 20 minutes | 30 minutes | |
Experimental details
| Sample | White adipocyte cell extract; 10 μg |
| Blocking | Incubated for 60 minutes with 5% skim milk |
| Detection |
ImmunoStar® LD manufactured by Fuji Film Wako, 15-minute exposure time ImmunoStar® LD is a trademark by Fuji Film Wako |
Data courtesy of Assistant Professor Tsuyoshi Goto and Dr. Masaomi Komori, Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University
Application 3
Flotillin-1, β-Actin and Nup98 proteins detection
| Antigen | Flotillin-1 | β-Actin | Nup98 | ||||
| Blotting image |  |
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| Antibody diluent | tTBS | Bullet ImmunoReaction Buffer | tTBS | Bullet ImmunoReaction Buffer | tTBS | Bullet ImmunoReaction Buffer | |
| Primary antibody | 60 min | 30 min | 60 min | 30 min | 60 min | 30 min | |
| Flotillin-1 antibody(C-7) (Santa Cruz #sc-133153) x 1,000 dilution | Human anti Human Actin Beta:HRP(Bio-Rad #HCA147P)x 1,000 dilution | Nup98 antibody(C-5) (Santa Cruz #sc-74578) x 1,000 dilution | |||||
| Secondary antibody | 60 min | 30 min |
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60 min | 30 min | ||
| Goat anti-Mouse IgG (H+L) Secondary Antibody [HRP] (Pre-adsorbed)(Novus #NB7574), x 40,000 dilution | Goat anti-Mouse IgG (H+L) Secondary Antibody [HRP] (Pre-adsorbed)(Novus #NB7574), x 40,000 dilution | ||||||
| Exposure time | 15 min | 15 min | 15 min | 5 min | 15 min | ||
Experimental details
| Sample |
Flotillin-1 from Hela cell extract, (left) 2 μg and (right) 0.4 μg β-Actin and Nup98 from Hela cell extract, (left) 10 μg and (right) 2 μg |
| Blocking | Incubated for 5 minutes with Bullet Blocking One |
| Detection | Chemi-Lumi One Super |
| Detector | LAS-300 High mode |
Transition to saving-time protocol
Transition chart
Performance comparison
| Antibody diluent |
Bullet ImmunoReaction Buffer |
tTBS | HIKARI |
A company |
A company（Protein-free） |
B company |
| Western blotting |  |
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| Primary antibody | 30 min | 60 min | ||||
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EGF Receptor (D38B1)XP® Rabbit mAb(CST #4267), x 1,000 dilution XP is a trademark by Cell Signaling Technology, Inc |
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| Secondary antibody | 30 min | 60 min | ||||
| Goat anti-Rabbit IgG (H+L) Secondary Antibody [HRP] (Pre-adsorbed)(Novus #NB7187), x 50,000 dilution | ||||||
Experimental details
| Sample | Hela cell extract, (left) 10 μg and (right) 2 μg |
| Blocking | Incubated for 5 minutes with Bullet Blocking One |
| Detection | Chemi-Lumi One Super manufactured by Nacalai, #02230; 3-minute exposure time |
| Detector | LAS-300 High mode |
Procedure
- Replace a current antibody diluent with Bullet ImmunoReaction Buffer
- Ab-Ag incubation time should be by half at a first trial; for a transition from overnight incubation protocol, 30–60-minute incubation time should be used.
- The instruction is downloadable through website, e-nacalai.
Blocking One (Instruction)