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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
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
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 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

-

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
Primary antibody 30 min 60 min

EGF Receptor (D38B1)XP® Rabbit mAb(CST #4267), x 1,000 dilution

XP is a trademark by Cell Signaling Technology, Inc

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.

Ordering Information

Product Cat.No. Storage Quantity Price(US$)  
Bullet ImmunoReaction Buffer 18439-85 2-8°C 500 ml 280.00 Buy