Anti-APC (Ab-5) Mouse mAb (CF11)

Purified mouse monoclonal antibody generated by immunizing mice with the specified immunogen and fusing splenocytes with SP2/0 mouse myeloma cells. Recognizes various full-length and truncated forms of APC.

Human

a recombinant protein consisting of the N-terminal 226 amino acids of APC

Immunoprecipitation (see comments and application references)

Please refer to vial label for lot-specific concentration.

In 50 mM sodium phosphate buffer, 0.2% gelatin.

Positive Control
HEK293, A-431, HCT116, or HT-1080 cells (full-length APC) or SW480 cells (truncated APC)

Smith, K.J., et al. 1993. Proc. Natl. Acad. Sci. USA90, 2846.
Su, L.-K., et al. 1993. Can. Res.53, 2728.
Boynton, R.F., et al. 1992. Proc. Natl. Acad. Sci. USA89, 3385.
D′Amico, D., et al. 1992. Cancer Res.52, 1996.
Fearon, E.R., and Jones, P.A. 1992. FASEB J.6, 2783.
Miyoshi, Y., et al. 1992. Proc. Natl. Acad. Sci. USA89, 4452.
Powell, S.M., et al. 1992. Nature359, 235.
Groden, J., et al. 1991. Cell66, 589.
Kinzler, K.W., et al. 1991. Science253, 661.
Nishisho, I., et al. 1991. Science253, 665.

Using this antibody various truncated and full lenghth forms of the APC protein have been observed in tumor cell lines and in lymphocytes obtained from FAP patients by immunoprecipiation. APC expression and turnover are very low making metabolic labelling difficult. APC immunocomplexes can be immunoprecipitated using Anti-APC (Ab-5) and detected in a immunoblot using Anti-APC (Ab-1), Cat. No. OP44 or Anti-APC (Ab-2), Cat. No. OP47L.



IMMUNOPRECIPITATION PROCEDURE



Can be used to immunoprecipitate full length and truncated APC protein APC. Precipitated APC proteins should then be visualized using APC (Ab-1) or APC (Ab-2) and the procedure provided with those products. APC expression and turnover are very low, therefore, immunoprecipitation of metabolically labeled lysates is not recommended.



Materials:



Equipment

• Rocking Platform



Solutions and Reagents:

• Anti-APC (Ab-5), Mouse mAb (CF11) Cat. No. OP62

• Protein A Sepharose swollen 1:1 in Wash Buffer

• Normal Mouse IgG

• Lysis Buffer: 100 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.5% NP-40 Alternative, 0.5 µg/ml Leupeptin, 1.0 µg/ml Pepstatin, 0.2 mM PMSF

• Wash Buffer: 100 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.5% NP-40 Alternative

• 2X Laemmli Sample Buffer: 120 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 200 mM DTT, 0.01% Bromophenol Blue



Procedure:

1) Lyse cells in lysis buffer. For adherent cells, lyse an 80-90% confluent 10 cm plate in 1.5 ml of buffer. Incubate at 4°C for 30 min with rocking. This will make enough lysate for 5 immunoprecipitations.

2) Transfer the lysate to a 1.5 ml microcentrifuge tube and centrifuge at 15,000 X g for 10 min at 4°C. Save supernatant–this is the lysate.

3) Pre-clear the 1.5 ml lysate with 10 µg normal mouse IgG (Cat. No. NI03) and 250 µl of Protein A sepharose solution (Cat. No. IP02). Incubate at 4°C for 60 min with continuous end-over-end mixing.

4) Pellet the Protein A Sepharose/mouse IgG complexes by centrifugation at 1000 X g for 1 min. Save the supernatant–this is the pre-cleared lysate.

5) Add 2 µg of APC (Ab-5) to 300 µl of the pre-cleared lysate and incubate at 4°C for 60 min with continuous end-over-end mixing.

6) Add 50 µl of Protein A sepharose and incubate at 4°C for 60 min with continuous end-over-end mixing.

7) Pellet the Protein A sepharose/immunocomplexes by centrifugation at 1000 X g for 1 min. Discard the supernatant.

8) Wash the Protein A sepharose/immunocomplex pellet 3 times with 1 ml of wash buffer.

9) Resuspend pellet in 50 µl of 2X Laemmli sample buffer and heat at 100°C for 10 min.

10) Detect APC proteins by immunoblotting as described for APC (Ab-1), Cat. No. OP44, or APC (Ab-2), Cat. No. OP47.

CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany

Toxicity: Standard Handling (A)