Bullet ImmunoReaction Buffer
When used for immunoblotting, Bullet ImmunoReaction Buffer may shorten antigen-antibody reaction time by half.
Feature
- 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 | ||||
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 Cytiva 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 minutes | 30 minutes | 60 minutes | 120 minutes | Overnight at 4°C |
---|---|---|---|---|---|
Secondary Ab incubation time | 15 minutes | 30 minutes | 60 minutes | 120 minutes | 60 minutes |
Bullet ImmunoReaction Buffer | |||||
0.1%-tTBS |
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 | |||
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 | |||||||
Antibody diluent | tTBS | Bullet ImmunoReaction Buffer | tTBS | Bullet ImmunoReaction Buffer | tTBS | Bullet ImmunoReaction Buffer | |
Primary antibody | 60 minutes | 30 minutes | 60 minutes | 30 minutes | 60 minutes | 30 minutes | |
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 minutes | 30 minutes | - | 60 minutes | 30 minutes | ||
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 minutes | 15 minutes | 15 minutes | 5 minutes | 15 minutes |
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 | ||||||
Primary antibody | 30 minutes | 60 minutes | ||||
EGF Receptor (D38B1)XP® Rabbit mAb(CST #4267), x 1,000 dilution XP is a trademark by Cell Signaling Technology, Inc. |
||||||
Secondary antibody | 30 minutes | 60 minutes | ||||
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.
Download
Bullet ImmunoReaction Buffer(PDF 2.52 MB)
Ordering information
Related Product : Bullet series
The Bullet series is designed to shorten the time required for western blotting, including SDS-PAGE, semi-dry blotting, blocking and Ab-Ag reaction as below figure.
Bullet series | Conventional method | |||||
---|---|---|---|---|---|---|
Western blotting | ||||||
Sample | Hela cell extract; 2-time dilution series started from 5 μg | |||||
Electrophoresis | Bullet PAGE One Precast Gel, 5-15%, 17 wells |
400 V | 10 mins. | Laemmli gel 10%, 18 wells |
200 V | 60 mins. |
Transfer | Bullet Semi-Dry Transfer One |
25 V | 10 mins. | Semi-dry transfer buffer for Western blotting |
10 V | 45 mins. |
Blocking | Bullet Blocking One | 5 mins. | 5% skim milk | 60 mins. | ||
Primary antibody GAPDH Antibody (Novous #NB300-322), x 10,000 dilution | Diluted with Bullet ImmunoReaction Buffer | 30 mins. | Diluted with 0.1% tTBS | 60 mins. | ||
Secondary antibody Goat anti-rabbit IgG(H+L) secondary antibody(Novous #MB7187), x 20,000 dilution | Diluted with Bullet ImmunoReaction Buffer | 30 mins. | Diluted with 0.1% tTBS | 60 mins. | ||
Detection | Chemi-Lumi One Super / 5-minute exposure time / LAS-3000 High mode | |||||
Total required time | About 2 hrs. | About 5 hrs. and 30 mins. |