Products

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 bc3fec7bb0114cc0567440e07152afe2.png cbc7aefb317b484368028870d18def88.png 5b0d0397eaf3c688f0b8409e7213cb3a.png ff96ccb0091a815e8c4cd99922c5cdd6.png
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 p2-Bullet15.png p2-Bullet30.png p2-Bullet60_1.png p2-Bullet120.png 2c01e0508ff609a209ffbc492be4cd86.png
0.1%-tTBS p2-tTBS15.png p2-tTBS30.png p2-tTBS60_1.png p2-tTBS120.png p2-tTBSon.png

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 p2-jisirei2-burotingzou_1.png
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 p2-flotllin-1-tTBS.png p2-flotllin-1-BIRB.png p2-actin-tTBS.png p2-actin-BIRB.png p2-Nup98-tTBS.png p2-Nup98-BIRB-5.png p2-Nup98-BIRB-30.png
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

Current antibody diluent.JPG

Performance comparison

Antibody diluent

Bullet

ImmunoReaction

Buffer

 tTBS HIKARI A company

A company

（Protein-free）

 B company
Western blotting p3-BIRB.png p3-tTBS_4.png p3-HIKARI.png p3-A.png p3-A-protein-free.png p3-B_4.png
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 Bufferpdf(PDF 2.52 MB)

Ordering information

Product Name Grade Storage Catalog Number PKG Size Online Catalog
Bullet ImmunoReaction Buffer SP Refrigerator 18439-85 500 mL e-Nacalai

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 p4-tyoukousokuka-Bullet.png p4-tyoukousokuka-jyuuraihou.png
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.

Ordering information

Category Product Name Grade Storage Catalog Number PKG Size Online Catalog
Precast gel Bullet PAGE One Precast Gel, 5-11%, 13wells SP Refrigerator 13077-04 10 sheets e-Nacalai
Bullet PAGE One Precast Gel, 5-11%, 17wells SP Refrigerator 13078-94 10 sheets e-Nacalai
Bullet PAGE One Precast Gel, 5-15%, 13wells SP Refrigerator 13079-84 10 sheets e-Nacalai
Bullet PAGE One Precast Gel, 5-15%, 17wells SP Refrigerator 13080-44 10 sheets e-Nacalai
Bullet PAGE One Precast Gel, 6%, 13wells SP Refrigerator 13081-34 10 sheets e-Nacalai
Bullet PAGE One Precast Gel, 6%, 17wells SP Refrigerator 13082-24 10 sheets e-Nacalai
Bullet PAGE One Precast Gel, 3%, 13wells SP Refrigerator 13083-14 10 sheets e-Nacalai
Bullet PAGE One Precast Gel, 3%, 17wells SP Refrigerator 13084-04 10 sheets e-Nacalai
Semi-dry transfer buffer Bullet Semi-dry Transfer One SP Room Temperature 15353-01 1 L e-Nacalai
Blocking reagent Bullet Blocking One for Western Blotting SP Refrigerator 13779-56 50 mL e-Nacalai
13779-14 200 mL e-Nacalai
13779-01 1 L e-Nacalai
Antibody diluent Bullet ImmunoReaction Buffer SP Refrigerator 18439-85 500 mL e-Nacalai
Gel staining solution Bullet CBB Stain One(Ready To Use) SP Refrigerator 13542-94 50 mL e-Nacalai
13542-65 500 mL e-Nacalai
13542-81 1 L e-Nacalai