371764 Gsα-Subunit, Recombinant, E. coli, Immunoblot Standard

371764
  
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      Recombinant Gsα-subunit expressed in E. coli. Partially purified from bacterial lysate by DEAE fractionation. Suitable for use as a positive control for immunoblotting. Use 10 µl per blot.
      Catalogue Number371764
      Brand Family Calbiochem®
      References
      ReferencesLinder, M.E. and Gilman, A.G. 1991. Methods Enzymol. 195, 202.
      Mumby, S., et al. 1988. J. Biol. Chem. 263, 2020.
      Mumby, S.M., et al. 1986. Proc. Natl. Acad. Sci. USA 83, 265.
      Tabor, S. and Richardson, C.C. 1985. Proc. Natl. Acad. Sci. USA 82, 1074.
      Product Information
      FormLiquid
      FormulationCrude DEAE fraction of bacterial lysate.
      PreservativeNone
      Applications
      Key Applications Immunoblotting (Western Blotting)
      Application NotesImmunoblotting (10 µl/lane)
      Application CommentsFor immunoblotting: 10 µg of purified plasma membranes from rat adipocytes and 10 µl of the Gsα standard were mixed with 2X sample buffer, boiled, and loaded into each lane. The proteins were resolved on a 12% acrylamide mini-gel (10 cm x 10 cm), transferred to PVDF membranes on a semi-dry blotter, and blocked in low detergent blotto (5% milk protein) for 1 hour. The blots were transferred to designated vessels containing the specific antibody diluted in low detergent blotto at 1:1000. Each blot was incubated on a rocker at room temperature with its specific antibody for 2 h, followed by 4 washes with low detergent blotto. Goat anti-rabbit IgG labeled with 125I (approximately 5µCi/µg IgG) was diluted to 500,000 cpm/ml in low detergent blotto, followed by several washes to remove milk proteins. The blots were exposed to X-ray film with intensifying screens. Other detection systems are also applicable.
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Standard Handling
      Storage ≤ -70°C
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
      Packaging Information
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      Gsα-Subunit, Recombinant, E. coli, Immunoblot Standard Certificates of Analysis

      TitleLot Number
      371764

      References

      Přehled odkazů
      Linder, M.E. and Gilman, A.G. 1991. Methods Enzymol. 195, 202.
      Mumby, S., et al. 1988. J. Biol. Chem. 263, 2020.
      Mumby, S.M., et al. 1986. Proc. Natl. Acad. Sci. USA 83, 265.
      Tabor, S. and Richardson, C.C. 1985. Proc. Natl. Acad. Sci. USA 82, 1074.
      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.

      Revision07-July-2008 RFH
      ApplicationImmunoblotting (10 µl/lane)
      DescriptionRecombinant Gsα-subunit expressed in E. coli. GTP-binding regulatory proteins (G-proteins) are heterotrimeric proteins associated with the cell membrane that functionally couple hormones to their receptor systems within the cell. These proteins link signaling pathways that regulate the synthesis of cyclic AMP, as well as other pathways, including activation of phospholipases and mobilization of intracellular calcium. The partially purified recombinant G-protein subunits, suitable for positive control for immunoblotting, can be used to check cross-reactivity in immunoblotting techniques. Plasmids for expression of this G-protein α-subunit were subcloned into plasmids and expressed in Escherichia coli by the method of Tabor and Richardson. The construction of the plasmids was described by Linder and Gilman.
      FormLiquid
      FormulationCrude DEAE fraction of bacterial lysate.
      Recommended reaction conditionsFor immunoblotting: 10 µg of purified plasma membranes from rat adipocytes and 10 µl of the Gsα standard were mixed with 2X sample buffer, boiled, and loaded into each lane. The proteins were resolved on a 12% acrylamide mini-gel (10 cm x 10 cm), transferred to PVDF membranes on a semi-dry blotter, and blocked in low detergent blotto (5% milk protein) for 1 hr. The blots were transferred to designated vessels containing the specific antibody diluted in low detergent blotto at 1:1000. Each blot was incubated on a rocker at room temperature with its specific antibody for 2 h, followed by 4 washes with low detergent blotto. Goat anti-rabbit IgG labeled with 125I (~5µCi/µg IgG) was diluted to 500,000 cpm/ml in low detergent blotto, followed by several washes to remove milk proteins. The blots were exposed to X-ray film with intensifying screens. Other detection systems are also applicable.
      PreservativeNone
      CommentsFor immunoblotting: 10 µg of purified plasma membranes from rat adipocytes and 10 µl of the Gsα standard were mixed with 2X sample buffer, boiled, and loaded into each lane. The proteins were resolved on a 12% acrylamide mini-gel (10 cm x 10 cm), transferred to PVDF membranes on a semi-dry blotter, and blocked in low detergent blotto (5% milk protein) for 1 hour. The blots were transferred to designated vessels containing the specific antibody diluted in low detergent blotto at 1:1000. Each blot was incubated on a rocker at room temperature with its specific antibody for 2 h, followed by 4 washes with low detergent blotto. Goat anti-rabbit IgG labeled with 125I (approximately 5µCi/µg IgG) was diluted to 500,000 cpm/ml in low detergent blotto, followed by several washes to remove milk proteins. The blots were exposed to X-ray film with intensifying screens. Other detection systems are also applicable.
      Storage ≤ -70°C
      Avoid freeze/thaw
      Do Not Freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
      Toxicity Standard Handling
      ReferencesLinder, M.E. and Gilman, A.G. 1991. Methods Enzymol. 195, 202.
      Mumby, S., et al. 1988. J. Biol. Chem. 263, 2020.
      Mumby, S.M., et al. 1986. Proc. Natl. Acad. Sci. USA 83, 265.
      Tabor, S. and Richardson, C.C. 1985. Proc. Natl. Acad. Sci. USA 82, 1074.