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

D031-3
Lot 027~
Page 1

MONOCLONAL ANTIBODY

Anti-Proton Pump (H,K-ATPase α subunit) mAb
Code No.
D031-3Clone
1H9Subclass
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
β subunit. The α subunit of H+, K+-ATPase has 10transmembrane domains and contains sites for ATP
binding and its acylphosphorylation, binding sites ofproton pump inhibitors, and sites responsible for ion
recognition.

SOURCE: This antibody was purified from ascites fluidusing protein A agarose. This hybridoma (clone 1H9) wasestablished by fusion of mouse myeloma cell Sp2/0-Ag14
with Balb/c nu/+ mouse splenocyte with neonatal
thymectomy-induced autoimmune gastritis.

FORMULATION: 100 µg IgG in 100 µL volume ofPBS containing 50% glycerol, pH 7.2. No preservative iscontained.

Quantity
100 µL

Concentration
1 mg/mL

SPECIES CROSS REACTIVITY:
Species

HumanMouse

Rat

BovineReactivity on WB++++INTENDED USE:

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

REFERENCES:

1) Becker, V., et al., World J. Gasteroenterol. 21, 982-987 (2015) [IHC]
2) Mimata, A., et al., Cancer Sci. 102, 942-950 (2011) [IHC]
3) Chandra, M., et al., Gasteroenterology 140, 857-867 (2011) [WB]
4) Katakai, T., et al., J. Immunol. 177, 7858-7867 (2006)
5) Kawamura, Y. I., et al., Cancer Res. 65, 6220-6227 (2005) [IHC]
6) Murphy, K., et al., Int. Immunol. 15, 269-277 (2003)
7) Laurie, K. L., et al., J. Immunol. 169, 2361-2367 (2002)
8) Kimura, T., et al., J. Biochem. 131, 923-932 (2002)
9) Kimura, T., et al., J. Biol. Chem. 277, 20671-20677 (2002)
10) Biondo, M., et al., J. Immunol. 166, 2090-2099 (2001)
11) Alderuccio, F., et al., Int. Immunol. 12, 343-352 (2000)
12) Jones, C. M., et al., Gastroenterology 101, 287-294 (1991)
13) Toh, B., et al., PNAS 87, 6418-6422 (1990)
14) Mori, Y., et al., Gastroenterology 97, 364-375 (1989)
Clone 1H9 is used in these references.

kDa 1 2

STORAGE: This antibody solution is stable for one year
from the date of purchase when stored at -20°C.

116
97

REACTIVITY: This antibody reacts with human,

66

APPLICATIONS:

45mouse, rat and bovine H , K -ATPase α subunit.
++

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.

isprovided

inthefollowing

Western blotting analysis ofproton pump on mouse stomachusing D031-3.

Lane 1: Boiled sample
Lane 2: Non-boiled sample

The 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

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.
URL https://ruo.mbl.co.jp e-mail support@mbl.co.jp

D031-3
Lot 027~
Page 2volume 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 at4°C with rotating for 30 minutes, then sonicate briefly(up to 10 seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°C
and transfer the supernatant to another tube. Measure theprotein concentration of the supernatant and add the Lysis
buffer to make 8 mg/mL solution.
3) Mix the sample with equal volume of Laemmli’s sample
buffer. Incubate at room temperature for 1 hour. Don’tboil the sample.
4) Load 10 µL 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 transfersystem (Transfer Buffer: 25 mM Tris, 190 mM glycine,20% methanol). 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 4°C.
7) Incubate the membrane with primary antibody diluted
with 1% skimmed milk (in PBS, pH 7.2) as suggested in
the APPLICATIONS for 1 hour at room temperature.
(The concentration of antibody to be used will depend on
the conditions.)
8) Wash the membrane with PBS-T [0.05% Tween-20 inPBS] (5 minutes x 3).
9) Incubate the membrane with 1:10,000 of Anti-IgG(Mouse) pAb-HRP (MBL, code no. 330) diluted with 1%skimmed milk (in PBS, pH 7.2) for 1 hour at roomtemperature.
10) Wash the membrane with PBS-T (5 minutes x 6).
11) Wipe excess buffer on the membrane, then incubate itwith appropriate chemiluminescence reagent for 1 minute.
Remove extra reagent from the membrane by dabbing
with paper towel, and seal it in plastic wrap.
12) Expose to an X-ray film in a dark room for 5 minutes.
Develop the film as usual. The condition for exposureand development may vary.

Immunohistochemical staining for paraffin-embedded
sections : SAB method

1) Deparaffinize the sections with Xylene 3 times for 3-5minutes 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 in PBS for 5 minutes each.
5) Remove the slides from PBS, wipe gently around eachsection and cover tissues with blocking buffer (20 mM
HEPES, 1% BSA, 135 mM NaCl, pH 7.2) 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 diluted
with blocking buffer as suggested 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 withHistostar™ (Ms + Rb) for Human tissue (MBL, code no.
8460). Incubate for 1 hour at room temperature. Wash asin step 9).
10) Visualize by reacting for 3-10 minutes with Histostar™
DAB Substrate Solution (MBL, code no. 8469). *DAB is asuspect carcinogen and must be handled with care. Always wear gloves.

11) Wash the slides in water for 5 minutes.
12) Counter stain in hematoxylin for 2 minutes, wash theslides 3 times in water for 5 minutes each, and thenimmerse the slides in PBS for 5 minutes. Dehydrate byimmersing in Ethanol 3 times for 3 minutes each,followed by immersing in Xylene 3 times for 3 minuteseach.
13) Now ready for mounting.
(Positive control for Immunohistochemistry; Humanstomach)

RELATED PRODUCTS
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Immunohistochemical detection ofproton pump on human stomachparaffin embedded section withD031-3.
(Positive control for Western blotting; Mouse stomach)

220201-6