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

D032-3HPage 1 of 2

MONOCLONAL ANTIBODY

Anti-Proton Pump (H,K-ATPase β subunit) mAb
Code No.
D032-3HClone
2B6Subclass
Mouse IgG1

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  subunit and a smaller but heavily glycosylated
 subunit. The  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.

BACKGROUND: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.

FORMULATION: 6 mL of prediluted antibody

Quantity
6 mL

Form
LiquidSPECIES CROSS REACTIVITY:
Species

Human

Mouse

Rat

BovineReactivity on WB++++REFERENCES:
1) Rieder, G., et al., J. Biol. Chem. 280, 4906-4912 (2005)
2) Murphy, K., et al., Int. Immunol. 15, 269-277 (2003)
3) Laurie, K. L., et al., J. Immunol. 169, 2361-2367 (2002)
4) Kimura, T., et al., J. Biochem. 131, 923-932 (2002)
5) Biondo, M., et al., J. Immunol. 166, 2090-2099 (2001)
6) Kimura, T., et al., J. Biol. Chem. 277, 20671-20677 (2002)
7) Asano, S., et al., J. Biol. Chem. 275, 8324-8330 (2000)
8) Lambrecht, N., et al., J. Biol. Chem. 275, 4041-4048 (2000)
9) Alderuccio, F., et al., Int. Immunol. 12, 343-352 (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) Toh, B., et al., PNAS 87, 6418-6422 (1990)
13) Mori, Y., et al., Gastroenterology 97, 364-375 (1989)
Clone 2B6 is used in these references.

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containing 0.09% NaN3.
*Azide may react with copper or lead in plumbing system toform explosive metal azides. Therefore, always flush plenty ofwater when disposing materials containing azide into drain.

STORAGE: This antibody solution is stable for 3 years
from the date of purchase when stored at 4°C.

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

APPLICATIONS:
Immunohistochemistry; Ready for use.
Detailed

procedure
PROTOCOL.

isprovidedinthefollowing

INTENDED USE:

Immunohistochemical detection ofproton pump on human stomachparaffin embedded section withD032-3H.

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

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

D032-3H
Page 2 of 2

PROTOCOL:
Immunohistochemical staining for paraffin-embedded
sections: SAB method
1) Deparaffinize the sections with Xylene 3 times for 5minutes each.
2) Wash the slides with Ethanol 3 times for 5 minutes each.
3) Wash the slides with PBS 3 times for 5 minutes each.
4) Remove the slides from PBS and cover each section with
3% H2O2 for 10 minutes at room temperature to blockendogenous peroxidase activity. Wash 2 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) 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 one or two drops ofAnti-Proton Pump (H,K-ATPase β subunit) mAb(D032-3H) (Ready for use).
7) Incubate the sections for 1 hour at room temperature.
8) Wash the slides 2 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 30 minutes at room temperature.
Wash as in step 8).
10) Visualize by reacting for 5 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; stomach)

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