For Research Use Only.
Not for use in diagnostic procedures.

D032-3
Lot 024~
Page 1

MONOCLONAL ANTIBODY

Anti-Proton pump/H,K-ATPase β subunit
Code No.
D032-3Clone
2B6

Subclass
Mouse IgG1

BACKGROUND:

Hydrogen-potassium
adenosine
triphosphatase (H+, K+-ATPase) belongs to a family ofP-type cation-transporting ATPases that also includes
Ca2+-ATPase and Na+, K+-ATPase. H+, K+-ATPase isresponsible for acid secretion into the stomach andcatalyzes electroneutral exchange of cytoplasmic hydrogenions and external potassium ions coupled with ATPhydrolysis. The enzyme is found in gastric parietal cells
where it is located in cytoplasmic vesicles or apical plasma
membranes of the secretory canaliculus. H+, K+-ATPase isa heterodimer consisting of a high molecular weightcatalytic α subunit and a smaller but heavily glycosylated βsubunit. The α subunit of H+, K+-ATPase has 10transmembrane domains and contains sites for ATP binding
and its acylphosphorylation, binding sites of proton pumpinhibitors, and sites responsible for ion recognition.

SOURCE: This antibody was purified from hybridoma(clone 2B6) supernatant using protein A agarose. Thishybridoma was established by fusion of mouse myeloma
cell Sp2/0-Ag14 with Balb/c nu/+ mouse splenocyte withneonatal thymectomy-induced autoimmune gastritis.

Quantity
100 µgConcentration
1 mg/mL

SPECIES CROSS REACTIVITY:
Species

Human

Mouse

Rat

BovineReactivity on WB++++REFERENCES:

1) Kaparakis, M., et al., Infect. Immun. 74, 3519-3529 (2006)
2) Vagin, O., et al., J. Biol. Chem. 280, 14741-14754 (2005)
3) Rieder, G., et al., J. Biol. Chem., 280, 4906-4912 (2005)4) Murphy, K., et al., Int. Immunol. 15, 269-277 (2003)
5) Laurie, K. L., et al., J. Immunol. 169, 2361-2367 (2002)
6) Biondo, M., et al., J. Immunol. 166, 2090-2099 (2001)
7) Alderuccio, F., et al., Int. Immunol. 12, 343-352 (2000)
8) Lambrecht, N., et al., J. Biol. Chem. 275, 4041-4048 (2000)
9) Asano, S., et al., J. Biol. Chem. 275, 8324-8330 (2000)
10) Asano, S., et al., J. Biol. Chem. 274, 22257-22265 (1999)
11) Stewart, L. A., et al., Glycobiology. 9, 601-606 (1999)
12) Jones, C. M., et al., Gasteroenterology 101, 287-294 (1991)
13) Toh, B., et al., PNAS. 87, 6418-6422 (1990)
14) Mori, Y., et al., Gasteroenterology 97, 364-375 (1989)
Clone 2B6 is used in these references.

FORMULATION: 100 µg IgG in 100 µL volume ofRELATED PRODUCTS:

STORAGE: This antibody solution is stable for one year

Clone 1H9 react with human, mouse, rat and bovine H+,
K+-ATPase α subunit.

PBS containing 50% glycerol, pH 7.2. No preservative iscontained.

from the date of purchase when stored at -20°C.

Please visit our website at https://ruo.mbl.co.jp/.

REACTIVITY: This antibody reacts with human, mouse,
rat and bovine H+, K+-ATPase β subunit.

APPLICATIONS:

Western blotting; 2-10 µg/mL
Immunoprecipitation; Not tested
It is reported that this monoclonal antibody can be usedin Immunoprecipitation in the reference 14).
Immunohistochemistry; 2-10 µg/mL
Immunocytochemistry; Not tested
Flow cytometry; Not tested
Detailed

procedure
PROTOCOLS.

isprovidedinthefollowing

INTENDED USE:

For Research Use Only. Not for use in diagnostic procedures.

URL https://ruo.mbl.co.jp e-mail support@mbl.co.jp

D032-3
Lot 024~
Page 2The descriptions of the following protocols are examples.
Each user should determine the appropriate condition.

PROTOCOLS:

SDS-PAGE & Western Blotting1) Wash the cells 3 times with PBS and suspend with 10
volume of cold Lysis buffer (50 mM Tris-HCl, pH 7.2,
250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol)containing appropriate protease inhibitors. Incubate it at
4oC with rotating for 30 minutes, then sonicate briefly (upto 10 seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4oC
and transfer the supernatant to another tube. Measure theprotein concentration of the supernatant and add the cold
Lysis buffer to make 8 mg/mL solution.
3) Mix the sample with equal volume of Laemmli’s sample
buffer.
4) Boil the samples for 3 minutes and centrifuge. Load 10µL of the sample per lane in a 1 mm thick
SDS-polyacrylamide gel for electrophoresis.
5) Blot the protein to a polyvinylidene difluoride (PVDF)
membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer
system (Transfer Buffer: 25 mM Tris, 190 mM glycine,
20% MeOH). See the manufacture's manual for precisetransfer procedure.
6) To reduce nonspecific binding, soak the membrane in
10% skimmed milk (in PBS, pH 7.2) for 1 hour at room
temperature, or overnight at 4oC.
7) Incubate the membrane with primary antibody diluted
with PBS, pH 7.2 containing 1% skimmed milk assuggest in the APPLICATIONS for 1 hour at room
temperature. (The concentration of antibody will dependon condition.)
8) Wash the membrane with PBS-T [0.05% Tween-20 inPBS] (5 minutes x 3 times).
9) Incubate
themembranewith
the1:10,000
HRP-conjugated anti-mouse IgG (MBL; code no. 330)
diluted with 1% skimmed milk (in PBS, pH 7.2) for 1
hour at room temperature.
10) Wash the membrane with PBS-T (10 minutes x 3 times).
11) Wipe excess buffer on the membrane, then incubate itwith appropriate chemiluminescence reagent for 1 minute.
12) Remove extra reagent from the membrane by dabbing
with paper towel, and seal it in plastic wrap.
13) Expose to an X-ray film in a dark room for 3 minutes.
14) Develop the film as usual. The condition for exposure anddevelopment may vary.
(Positive control for Western blotting; mouse stomach)

Immunohistochemical detection of
proton pump on human stomachparaffin embedded section withD032-3.
Immunohistochemical staining for paraffin-embedded
sections: SAB method

1) Deparaffinize the sections with Xylene 3 times for 3-5
minutes each.
2) Wash the slides with Ethanol 3 times for 3-5 minuteseach.
3) Wash the slides with PBS 3 times for 3-5 minutes each.
4) Remove the slides from the citrate buffer and cover eachsection with 3% H2O2 for 10 minutes at room temperature
to block endogenous peroxidase activity. Wash 3 times inPBS for 5 minutes each.
5) Remove the slides from PBS, wipe gently around eachsection and cover tissues with Protein Blocking Agent(Ultratech HRP Kit; MBL, code no. IM-2391) for 5
minutes to block non-specific staining. Do not wash.
6) Tip off the blocking buffer, wipe gently around eachsection and cover tissues with primary antibody dilutedwith PBS containing 1% BSA as suggest in theAPPLICATIONS.
7) Incubate the sections for 1 hour at room temperature.
8) Wash the slides 3 times in PBS for 5 minutes each.
9) Wipe gently around each section and cover tissues withPolyvalent Biotinylated Antibody (Ultratech HRP Kit).
Incubate for 15 minutes at room temperature. Wash as instep 9).
10) Wipe gently around each section and cover tissues with
Streptavidin-Peroxidase (Ultratech HRP Kit). Incubate for
15 minutes at room temperature. Wash as in step 9).
11) Visualize by reacting for 10-20 minutes with substratesolution containing 7.5 mg DAB, 40 µL of 30% H2O2 in
150 mL PBS. *DAB is a suspect carcinogen and must be handledwith care. Always wear gloves.

12) Wash the slides in water for 5 minutes.
13) Counter stain in hematoxylin for 1 minute, wash the slides
3 times in water for 5 minutes each, and then immerse theslides in PBS for 5 minutes. Dehydrate by immersing in
Ethanol 3 times for 3 minutes each, followed byimmersing in Xylene 3 times for 3 minutes each.
14) Now ready for mounting.
(Positive controls for Immunohistochemistry; human stomach)
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