Egf Mouse Monoclonal Antibody [Clone ID: E5]

CAT#: AM32087SU-N

Egf mouse monoclonal antibody, clone E5, Supernatant

Specifications

Product Data

• Clone Name: E5
• Applications: ELISA, IHC
• Recommended Dilution: ELISA.
  Spot Blots.
  Immunohistochemistry on Fixed Frozen Sections: 1/20.
  Immunohistochemistry on Paraffin Sections of Salivary glands (see Protocols for more details.)
• Reactivities: Mouse
• Host: Mouse
• Isotype: IgG1
• Clonality: Monoclonal
• Specificity:

  The antibody reacts with Mouse EGF in ELISA (10 ng detectable) and in Spot Blots (1 ng detectable).
  In Immunohistochemistry the antibody reacts with Mouse salivary glands.

• Formulation: State: Supernatant
  State: Tissue Culture Supernatant
  Stabilizer: 1.0% BSA
  Preservative: 20 mM Sodium Azide
• Concentration: lot specific
• Conjugation: Unconjugated
• Storage: Store the antibody undiluted at 2-8°C for one month or (in aliquots) at -20°C for longer.
  Avoid repeated freezing and thawing.
• Stability: Shelf life: one year from despatch.
• Gene Name: epidermal growth factor
• Database Link:
  Entrez Gene 13645 Mouse
  UniProt ID P01132
• Background: Epidermal growth factor (EGF) has a profound effect on the differentiation of specific cells in vivo and is a potent mitogenic factor for a variety of cultured cells. The EGF precursor is believed to exist as a membrane-bound molecule which is proteolytically cleaved to generate the 53-amino acid peptide hormone that stimulates cells to divide. EGF exerts its actions by binding to the EGFR, a 170 kDa protein.
  Epidermal growth factor (EGF) is a key growth factor regulating cell survival. Through its binding to cell surface receptors, EGF activates an extensive network of signal transduction pathways that include activation of the PI3K/AKT, RAS/ERK and JAK/STAT pathways. Because of its key role in driving the proliferation of cells, EGFR is a target of several anti-cancer drugs currently in development.
• Synonyms: Urogastrone, Epidermal growth factor, URG, HOMG4
• Note: Protocol:

  Immunoblotting/Spotting
  1. Homogenize samples in sample buffer containing 50mM Tris-HCL (pH 6.8), 0.01% SDS, 0.6mM glycerol, and 0.33 M ß-mercaptoethanol.
  2. Heat for 5 min. at 100°C. Cool at room-temperature.
  3. Centrifugate the samples at 10,000 x g for 5 min.
  4. Samples of purified mEGF with and without prior heatening in ß-mercaptoethanol were subjected to PAGE according to Maizel
  5. After electrophoresis, the gels were soaked for 30 min in H₂O to reduce SDS concentration and then blotted on nitrocellulose paper according to Towbin et al (J. electrophoretic transfer of proteins from polyacrylamide gels to nitro cellulose sheets: procedure and some applications. Proc. Natl Acad. Sci USA 1979;76:4350), with voltage gradient of 5V/cm for periods ranging from 15 min. - 2 hr.
  6. After electrotransfer of proteins to nitrocellulose paper, the paper was baked overnight at 60°C and the remaining protein binding sites were blocked with 3% ovalbumin in PBS for at least 1 hr.
  7. Strips of the paper were then incubated with hybridoma culture medium and were developed with RAM-HPO followed by DAB + H₂O.
  8. Control incubations were done with SP2/0 culture medium For analysis of reactions with other proteins containing EGF-like sequences, these proteins were spotted on nitrocellulose strips, which were then allowed to dry: Spots containing such proteins were not baked at 60°C.
  
  Indirect Immunoperoxidase Staining On Frozen Sections
  1. 4 to 6 micron thick sections should be used.
  2. Sections are thawed, 1-2 hours at room temperature.
  3. Tissue is fixed in acetone, 10 minutes.
  4. Wash with PBS, 2 x 3 minutes.
  5. Incubate with monoclonal antibody (diluted in PBS), 1-2 hours at room temperature.
  6. Wash with PBS, 3 x 3 minutes.
  7. Incubate with peroxidase labeled second antibody, 30-60 minutes at room temperature.
  8. Wash with PBS, 3 x 3 minutes.
  9. Stain with diaminobenzidin (DAB) solution 10 minutes at room temperature.
  10. Wash with running tap water, 3 minutes.
  11. Counterstain with Mayer's hematoxylin, 2 minutes.
  12. Wash with running tap water, 5 minutes.
  13. Dehydrate with increasing solution of ethanol; 50%, 70%, 96%, absolute, 3 minutes each.
  14. Clear with xylol, 3 x 3 minutes.
  15. Mount with mounting medium (e.g. malinol).
  
  Indirect Immunoperoxidase Staining On Formalin-Fixed And Paraffin Embedded Tissues
  1. 4 micron thick sections should be used.
  2. Dewax in Xylol, 3 x 3 minutes.
  3. Rehydrate in decreasing grades of ethanol:absolute, 96%, 70%, 50%, 3 minutes each.
  4. Block endogenous peroxidase activity with freshly made 0.3% H₂O₂ in methanol, 20 minutes.
  5. Wash with PBS, 3 x 3 minutes.
  Only if trypinsination is required
  5a. Incubate sections with 0.1% Trypsin in 0.1% CaCl₂ pH 7.6 for 10 minutes at room temperature.
  5b. Wash with PBS, 3 x 3 minutes.
  6. Cover the sections with 20% normal rabbit serum in PBS or normal human serum and incubate overnight in a humidity chamber at room temperature to reduce non specific background staining.
  7. Decant 20% normal rabbit serum.
  8. Incubate with monoclonal antibody (diluted in PBS), 1-2 hours at room temperature.
  9. Wash with PBS, 3 x 3 minutes.
  10. Incubate with peroxidase labeled second antibody, 30-60 minutes at room temperature.
  11. Wash with PBS, 3 x 3 minutes.
  12. Stain with diaminobensidin (DAB) solution, 10 minutes at room temperature. A stock solution of 0.5% DAB in 0.5 DAB in 0.5M Tris/HCl (pH7.4) can be made and stored frozen in the dark. Before use a quantity needed for staining can be thawed and diluted 10x with water. The diluted DAB solution should be filtrated. Just before use H2O2 must be added to a final concentration of 0.01%.
  13. Wash with running tap water, 3 minutes.
  14. Counterstain with Mayer's hematoxylin, 2 minutes.
  15. Wash with running tap water, 2 minutes.
  16. Dehydrate with increasing solutions of ethanol:50%, 70%, 96%, absolute, 3 minutes each.
  17. Clear with xylol, 3 x 3 minutes.
  18. Mount with mounting medium (e.g. malinol).