D050-3
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For Research Use Only.
Not for use in diagnostic procedures.

MONOCLONAL ANTIBODY

CD29/1-Integrin
Code No.
D050-3Clone
AG89

Subclass
Mouse IgG1

BACKGROUND: The integrin family of adhesion
molecules participate in important cell-cell andcell-extracellular matrix interactions in a diverse range of
biological processes. Integrins are heterodimers consistingof a subunit and  subunit. Both and  subunits aretransmembrane proteins with large extracellular domains(>100 kDa for subunit and >75 kDa for  subunit) thatinteract with extracellular matrix proteins and relatively
small cytoplasmic domains (50 amino acids or less, except
for the 4 subunit) that interact with cytoskeletal proteins.
The adhesiveness of integrins is dynamically regulated inresponse to cytoplasmic signals, termed “inside-out”
signaling. It has been reported that, upon ligand binding,integrins regulate many intracellular signaling pathways
that involve cytoplasmic alkalization, intracellular Ca2+fluctuation, inositol lipid metabolism, protein kinase C,
MAP kinase and phosphatidyl inositol kinase. Anti-integrin
monoclonal antibody, AG89, reacts with CD29/integrin 1
chain regardless of the subunit. AG89 can recognize restingsite 1 integrin on the cells, but the reactivity is increased~2-fold upon integrin activation by anti-activating 1
antibodies and ~3-fold by Mn2+. Furthermore, occupationof the ligand-binding pocket by a soluble ligand (RGD
peptide for 51 and CS-1 peptide for 41) resulted in
maximum binding of AG89. The epitope for AG89 lies
within residues 426-587.

SOURCE: This antibody was purified from mouse ascites
fluid using protein A agarose. This hybridoma wasestablished by fusion of mouse myeloma cell P3U1 with
Balb/c mouse splenocyte immunized with G-361 humanmelanoma cell line.

FORMULATION: This antibody is lyophilized form.
Prepare a stock solution by dissolving the lyophilized
antibody in 100 L of distilled water. After reconstitution,
the IgG concentration should be 1 mg/mL in PBS (pH 7.2)/1% sucrose. No preservative contained.

STORAGE: This antibody is stable for one year from thedate of shipment when stored at 4oC. After reconstitution,
avoid repeated freezing and thawing. For storage, prepare
appropriate aliquots and freeze them at -20oC.

REACTIVITY: This antibody reacts with human
1-Integrin on Flow cytometry.

Quantity
100 gForm
Lyophilized

APPLICATIONS:

Western blotting; 10 g/mL for chemiluminescence
detection system
Immunoprecipitation; Not tested
It is reported that this monoclonal antibody can be usedin Immunoprecipitation in the reference number 4).
Immunohistochemistry; Not tested
Immunocytochemistry; Not tested
Flow cytometry; 10 g/mL (final concentration)
Detailed

procedure
PROTOCOLS.

isprovidedinthefollowing

SPECIES CROSS REACTIVITY:
Species
Cells
Reactivity on FCM

Human
MOLT-4,
JurkatMouse

Rat

Not Tested

Not Tested+REFERENCES:
1) Nishiuchi. R., et al. PNAS 102, 1939-1944 (2005)
2) Shi, Q., et al. Mol. Biol. Cell 14, 4306-4315 (2003)
3) García, J. A., et al. J. Biol. Chem. 273, 34710-34715 (1998)
4) Tsuchida. J., et al. J. Cell Sci. 111, 1759-1766 (1998)
5) Takagi. J., et al. J. Biochem. 121, 914-921 (1997)
6) O'Toole, T.E., et al. J. Cell Biol. 124, 1047-1059 (1994)
Clone AG89 is used in the reference number 1) - 5).

RELATED PRODUCTS:
D050-5
K0099-3
D202-3
D202-4
M100-3
M100-4
M100-6
M109-3
K0046-3
K0046-5
K0047-3

PE labeled CD29/1-Integrin (AG89)
Anti-MIBP (5B4.7)
mouse CD11b (1C4)FITC labeled mouse CD11b (1C4)
mouse CD11c (223H7)
FITC labeled mouse CD11c (223H7)
Biotin labeled mouse CD11c (223H7)
mouse CD61 (1-55-4)
Anti-mouse Integrin 7 (3C12)
PE labeled anti-mouse Integrin 7 (3C12)
Anti-mouse Integrin 7 (6A11)

PROTOCOLS:SDS-PAGE & Western Blotting

INTENDED USE:
For Research Use Only. Not for use in diagnostic procedures.

1) Wash the cells 3 times with PBS and suspend with 10
volume of cold Lysis buffer (50 mM Tris-HCl, pH 7.2,

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D050-3
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250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol)containing appropriate protease inhibitors. Incubate it at4oC 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 samplebuffer.
4) Boil the samples for 3 minutes and centrifuge. Load 10
L 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% 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.

concentration of antibody will depend on condition).
6) Add 1 mL of the washing buffer followed bycentrifugation at 500 x g for 1 minute at room temperature.
Remove supernatant by careful aspiration.
7) Add 30 L of 1:100 FITC conjugated anti-mouse IgG(MBL; code no. IM-0819) diluted with the washing buffer.
Mix well and incubate for 15 minutes at roomtemperature.
8) Add 1 mL of the washing buffer followed bycentrifugation at 500 x g for 1 minute at room temperature.
Remove supernatant by careful aspiration.
9) Resuspend the cells with 500 L of the washing buffer
and analyze by a flow cytometer.
(Positive controls for Flow cytometry; MOLT-4, Jurkat)

Flow cytometric analysis of CD29/1 Integrin
expression on MOLT-4 (left) and Jurkat (right).
Open histograms indicate the reaction of isotypic
control to the cells. Shaded histograms indicate
the reaction of D050-3 to the cells.

Flow cytometric analysis for floating cells
We usually use Fisher tubes or equivalents as reaction tubes
for all step described below.
1) Wash the cells 3 times with washing buffer [PBS
containing 2% fetal calf serum (FCS) and 0.1% NaN3].
2) Resuspend the cells with washing buffer (5x106 cells/mL).
3) Add 50 L of the cell suspension into each tube, andcentrifuge at 500 x g for 1 minute at room temperature(20~25oC). Remove supernatant by careful aspiration.
4) Add 10 L of normal goat serum containing 1 mg/mL
normal human IgG and 0.1% NaN3 to the cell pellet aftertapping. Mix well and incubate for 5 minutes at roomtemperature.
5) Add 30 L of primary antibody diluted as suggest in theAPPLICATIONS to cell pellet and gently mix thenincubate for 30 minutes at room temperature (The070427-2.1