Nacalai USA, Inc. | Product | 30 min Antigen-Antibody Reaction: Bullet ImmunoReaction Buffer

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
Primary antibody	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
Secondary antibody	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.

Downloads

Blocking One (Instruction)

Reference

Hossin, Abu Yousuf, et al. "Syringin: A Phenylpropanoid Glycoside Compound in Cirsium brevicaule A. GRAY Root Modulates Adipogenesis." Molecules 26.6 (2021): 1531.

Ordering Information

Product	Cat.No.	Storage	PKG Size	Price(US$)
Bullet ImmunoReaction Buffer	18439-85	2-8°C	500 ml	294.00	Buy