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OP44 Anti-APC (Ab-1) Mouse mAb (FE9)

Anti-APC (Ab-1), mouse monoclonal, clone FE9, recognizes full length APC (p300) in HCT116 cells and truncated APC (p147) in SW480 cells. It is validated for Western botting.

Anti-APC (Ab-1) Mouse mAb (FE9) MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms: Anti-Adenomatous Polyposis Coli

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OP44
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Overview

Replacement Information

Key Spec Table

Species ReactivityHostAntibody Type
H, M, RMMonoclonal Antibody

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      Description
      OverviewRecognizes full length APC (p300) in HCT116 cells and truncated APC (p147) in SW480 cells.
    • Antibody Target Gene Symbol: APC
    • Target Synonym: AI047805, Apc7, AU020952, AW124434, BTPS2, DP2, DP2.5, DP3, Familial adenomatous polyposis, FAP, GS, Min, RATAPC
    • Entrez Gene Name: adenomatous polyposis coli
    • Hu Entrez ID: 324 (Related Antibodies: OP80, ST1150, OP62, OP47L)
    • Mu Entrez ID: 11789
    • Rat Entrez ID: 24205
      		•
      Catalogue NumberOP44
      Brand Family Calbiochem®
      SynonymsAnti-Adenomatous Polyposis Coli
      References
      ReferencesKoetsier, P. A., et al. 1993. BioTechniques 15, 258.
      Smith, K. J., et al. 1993. Proc. Natl. Acad. Sci., USA 90, 2846.
      Su, L.-K., et al. 1993. Can. Res. 53, 2728.
      Boynton, R. F., et al. 1992. Proc. Natl. Acad. Sci. USA 89, 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. USA 89, 4452.
      Powell, S. M., et al. 1992. Nature 359, 235.
      Groden, J., et al. 1991. Cell 66, 589.
      Kinzler, K. W., et al. 1991. Science 253, 661.
      Nishisho, I., et al. 1991. Science 253, 665.
      Product Information
      FormLiquid
      FormulationIn 50 mM sodium phosphate buffer, pH 7.5, 0.2% gelatin.
      Positive controlHCT116 cells for p300, SW480 cells for truncated APC (p147)
      Preservative≤0.1% sodium azide
      Quality LevelMQ100
      Applications
      Key Applications Immunoblotting (Western Blotting)
      Application NotesImmunoblotting (1 µg/ml, see comments)
      Application CommentsFull-length p300APC can be observed by western blot and various truncated forms of the APC protein have been observed in tumors obtained from sporadic colorectal cancer patients and in lymphocytes obtained from FAP patients. SW480 cells, which contain a mutation at codon 1338 of APC and express a truncated APC protein of 147 kDa, and HCT116 cells, which express full length APC, are available from ATCC for use as positive controls in western blotting with APC (Ab-1). Antibody should be titrated for optimal results in indivual systems.

      IMMUNOBLOTTING PROCEDURE

      Materials:
      1. Equipment
      Electrophoresis apparatus and Rocker platform

      2. Reagents and Solutions
      • Anti-APC (Ab-1) Mouse mAb (FE9), Cat. No. OP44
      • Secondary antibody (e.g. Goat Anti-Mouse IgG, H & L chain specific Peroxidase conjugate, Cat. No. 401215)
      • Chemiluminescent detection system
      • 1x Laemmli Sample Buffer: 60 mM Tris-Cl, pH 6.8, 2% SDS, 10% Glycerol, 100 mM DTT, 0.001% Bromophenol Blue
      • Laemmli Running Buffer: 25 mM Tris base, 192 mM Glycine, 0.1% SDS
      • 3% Low Melt Agarose (i.e. SeaPlaque from FMC) in TBE/0.1% SDS: TBE: 89 mM Tris base, 89 mM Boric acid, 2 mM EDTA pH 8.0
      • Tris-buffered saline (TBS) pH 7.6: 20 mM Tris base, 137 mM NaCl, pH to 7.6 with HCl
      • TBS/0.04% SDS
      • TBS/0.1% Tween®-20 detergent (TBST)
      • 10% Non-fat Dry Milk in TBST
      • 5% Non-fat Dry Milk in TBST

      Agarose Gel and Capillary Blotting Procedures:
      1) Lyse cells in 1x Laemmli Sample Buffer. For adherent cells, lyse a 10 cm plate that is 80 to 90% confluent in 2 ml of sample buffer added directly to cells on the plate. Final protein concentration is approximately 1 mg/ml.

      2) Electrophorese 50-100 µg of total lysate protein on a 3% low melt agarose gel made in TBE/0.1% SDS. Use a vertical polyacrylamide gel apparatus with 1 mm spacers. Pour a 1 cm 15% acrylamide plug. Prewarm the gel apparatus to 37°C before pouring the agarose gel. Pour the molten agarose while it is still 65° to 70°C. There is no stacking gel. Use Laemmli running buffer. Run the gel at 15V per centimeter until the proteins in the 40 kDa to 60 kDa range have migrated off the bottom of the gel.

      3) Transfer the proteins overnight onto a PVDF membrane by downward capillary transfer as outlined below using TBS/0.04% SDS as the transfer buffer:

      a. On a 2 inch stack of blotting paper or paper towels, place three pieces of gel-sized Whatman 3 MM chromatography paper with the top piece pre-soaked in transfer buffer.

      b. Place a pre-soaked, gel-sized piece of PVDF membrane on top of the Whatman papers.

      c. Place the gel on top of the membrane and cover the exposed papers with plastic wrap.

      d. Place two gel-sized pieces of pre-soaked Whatman 3MM paper on top of the gel.

      e. On opposite sides of the blotting stack place a tray containing transfer buffer.

      f. Lay two pre-soaked, long pieces of Whatman paper across the top of the gel set up so that the ends are submerged in the trays containing transfer buffer.

      g. Cover the entire set-up with plastic wrap and allow the transfer to go overnight.

      4) After overnight transfer, mark the position of protein standards on the PVDF membrane with a pencil. The membrane is then incubated for 1 h in 10% non-fat dry milk in TBST at room temperature with rocking. Use about 1 ml of solution per cm2 of membrane.

      5) Incubate the membrane with 1 µg/ml of APC antibody in 5% non-fat dry milk in TBST for 2 h at room temperature with rocking.

      6) Wash the membrane 3 times, 10 min each, in TBST at room temperature with rocking.

      7) Incubate the membrane with 30 ng/ml of HRP conjugated goat anti-mouse IgG heavy and light chain antibody in 5% non-fat dry milk in TBST at room temperature for 1 h.

      8) Wash the membrane 6 times, 15 min each, in TBST at room temperature with rocking.

      9) Develop the membrane using chemiluminescent detection reagents according to manufacturers instructions.

      10) Expose the membrane to film for 30 min. Adjust subsequent exposure times (from 30 s up to 24 h) as necessary.
      Biological Information
      Immunogena synthetic peptide corresponding to the N-terminal 35 amino acids of APC
      CloneFE9
      HostMouse
      IsotypeIgG₁
      Species Reactivity
      • Human
      • Mouse
      • Rat
      Antibody TypeMonoclonal Antibody
      Concentration Label Please refer to vial label for lot-specific concentration
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Standard Handling
      Storage +2°C to +8°C
      Do not freeze Yes
      Packaging Information
      Transport Information
      Supplemental Information
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      OP44-100UG 04055977227130

      Documentation

      Anti-APC (Ab-1) Mouse mAb (FE9) SDS

      Title

      Safety Data Sheet (SDS) 

      Anti-APC (Ab-1) Mouse mAb (FE9) Certificates of Analysis

      TitleLot Number
      OP44

      References

      Reference overview
      Koetsier, P. A., et al. 1993. BioTechniques 15, 258.
      Smith, K. J., et al. 1993. Proc. Natl. Acad. Sci., USA 90, 2846.
      Su, L.-K., et al. 1993. Can. Res. 53, 2728.
      Boynton, R. F., et al. 1992. Proc. Natl. Acad. Sci. USA 89, 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. USA 89, 4452.
      Powell, S. M., et al. 1992. Nature 359, 235.
      Groden, J., et al. 1991. Cell 66, 589.
      Kinzler, K. W., et al. 1991. Science 253, 661.
      Nishisho, I., et al. 1991. Science 253, 665.
      Data Sheet

      Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

      Revision10-May-2010 RFH
      SynonymsAnti-Adenomatous Polyposis Coli
      ApplicationImmunoblotting (1 µg/ml, see comments)
      DescriptionProtein G purified mouse monoclonal antibody generated by immunizing mice with the specified immunogen and fusing splenocytes with SP40 cells. Recognizes the ~300 kDa APC protein as well as a variety of truncated forms.
      BackgroundColon cancer generally occurs sporadically although there is clear genetic predisposition in some families. Familial Adenomatous Polyposis Coli (FAP) is an autosomal dominantly inherited disorder that exhibits 80 to 100% penetrance and predisposes to colon cancer. The gene responsible for FAP has been assigned to chromosome 5q21, a region that is often lost in tumors from patients with sporadic colorectal cancer. Recently, a gene called APC, for adenomatous polyposis coli, from the 5q21 locus was isolated and shown to contain mutations in DNA prepared from tumors of patients with sporadic colorectal cancer and in germ line DNA and tumor DNA from FAP patients. In 79 unrelated patients with FAP, mutations in APC were found in 67% of the cases; and 49 of the 53 mutations were either small deletions/insertion or were point mutations that introduced stop codons into the reading frame. Furthermore, APC mutations have been found in the earliest tumors that could be analyzed (0.5 cm diameter) and the mutation frequency remained constant as tumors progressed from benign to malignant stages. Analysis of the APC protein demonstrates that inactivating mutations result in stable truncated proteins that can be identified in colon tumors from both FAP and sporadic colon cancer patients as well as in constitutional cells of FAP patients. Loss of heterozygosity at the APC locus has also been reported for esophageal cancer and lung cancer.
      HostMouse
      Immunogena synthetic peptide corresponding to the N-terminal 35 amino acids of APC
      CloneFE9
      IsotypeIgG₁
      Specieshuman, mouse, rat
      Positive controlHCT116 cells for p300, SW480 cells for truncated APC (p147)
      FormLiquid
      FormulationIn 50 mM sodium phosphate buffer, pH 7.5, 0.2% gelatin.
      Concentration Label Please refer to vial label for lot-specific concentration
      Preservative≤0.1% sodium azide
      CommentsFull-length p300APC can be observed by western blot and various truncated forms of the APC protein have been observed in tumors obtained from sporadic colorectal cancer patients and in lymphocytes obtained from FAP patients. SW480 cells, which contain a mutation at codon 1338 of APC and express a truncated APC protein of 147 kDa, and HCT116 cells, which express full length APC, are available from ATCC for use as positive controls in western blotting with APC (Ab-1). Antibody should be titrated for optimal results in indivual systems.

      IMMUNOBLOTTING PROCEDURE

      Materials:
      1. Equipment
      Electrophoresis apparatus and Rocker platform

      2. Reagents and Solutions
      • Anti-APC (Ab-1) Mouse mAb (FE9), Cat. No. OP44
      • Secondary antibody (e.g. Goat Anti-Mouse IgG, H & L chain specific Peroxidase conjugate, Cat. No. 401215)
      • Chemiluminescent detection system
      • 1x Laemmli Sample Buffer: 60 mM Tris-Cl, pH 6.8, 2% SDS, 10% Glycerol, 100 mM DTT, 0.001% Bromophenol Blue
      • Laemmli Running Buffer: 25 mM Tris base, 192 mM Glycine, 0.1% SDS
      • 3% Low Melt Agarose (i.e. SeaPlaque from FMC) in TBE/0.1% SDS: TBE: 89 mM Tris base, 89 mM Boric acid, 2 mM EDTA pH 8.0
      • Tris-buffered saline (TBS) pH 7.6: 20 mM Tris base, 137 mM NaCl, pH to 7.6 with HCl
      • TBS/0.04% SDS
      • TBS/0.1% Tween®-20 detergent (TBST)
      • 10% Non-fat Dry Milk in TBST
      • 5% Non-fat Dry Milk in TBST

      Agarose Gel and Capillary Blotting Procedures:
      1) Lyse cells in 1x Laemmli Sample Buffer. For adherent cells, lyse a 10 cm plate that is 80 to 90% confluent in 2 ml of sample buffer added directly to cells on the plate. Final protein concentration is approximately 1 mg/ml.

      2) Electrophorese 50-100 µg of total lysate protein on a 3% low melt agarose gel made in TBE/0.1% SDS. Use a vertical polyacrylamide gel apparatus with 1 mm spacers. Pour a 1 cm 15% acrylamide plug. Prewarm the gel apparatus to 37°C before pouring the agarose gel. Pour the molten agarose while it is still 65° to 70°C. There is no stacking gel. Use Laemmli running buffer. Run the gel at 15V per centimeter until the proteins in the 40 kDa to 60 kDa range have migrated off the bottom of the gel.

      3) Transfer the proteins overnight onto a PVDF membrane by downward capillary transfer as outlined below using TBS/0.04% SDS as the transfer buffer:

      a. On a 2 inch stack of blotting paper or paper towels, place three pieces of gel-sized Whatman 3 MM chromatography paper with the top piece pre-soaked in transfer buffer.

      b. Place a pre-soaked, gel-sized piece of PVDF membrane on top of the Whatman papers.

      c. Place the gel on top of the membrane and cover the exposed papers with plastic wrap.

      d. Place two gel-sized pieces of pre-soaked Whatman 3MM paper on top of the gel.

      e. On opposite sides of the blotting stack place a tray containing transfer buffer.

      f. Lay two pre-soaked, long pieces of Whatman paper across the top of the gel set up so that the ends are submerged in the trays containing transfer buffer.

      g. Cover the entire set-up with plastic wrap and allow the transfer to go overnight.

      4) After overnight transfer, mark the position of protein standards on the PVDF membrane with a pencil. The membrane is then incubated for 1 h in 10% non-fat dry milk in TBST at room temperature with rocking. Use about 1 ml of solution per cm2 of membrane.

      5) Incubate the membrane with 1 µg/ml of APC antibody in 5% non-fat dry milk in TBST for 2 h at room temperature with rocking.

      6) Wash the membrane 3 times, 10 min each, in TBST at room temperature with rocking.

      7) Incubate the membrane with 30 ng/ml of HRP conjugated goat anti-mouse IgG heavy and light chain antibody in 5% non-fat dry milk in TBST at room temperature for 1 h.

      8) Wash the membrane 6 times, 15 min each, in TBST at room temperature with rocking.

      9) Develop the membrane using chemiluminescent detection reagents according to manufacturers instructions.

      10) Expose the membrane to film for 30 min. Adjust subsequent exposure times (from 30 s up to 24 h) as necessary.
      Storage +2°C to +8°C
      Do Not Freeze Yes
      Toxicity Standard Handling
      ReferencesKoetsier, P. A., et al. 1993. BioTechniques 15, 258.
      Smith, K. J., et al. 1993. Proc. Natl. Acad. Sci., USA 90, 2846.
      Su, L.-K., et al. 1993. Can. Res. 53, 2728.
      Boynton, R. F., et al. 1992. Proc. Natl. Acad. Sci. USA 89, 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. USA 89, 4452.
      Powell, S. M., et al. 1992. Nature 359, 235.
      Groden, J., et al. 1991. Cell 66, 589.
      Kinzler, K. W., et al. 1991. Science 253, 661.
      Nishisho, I., et al. 1991. Science 253, 665.