371768 G-Protein, βγ-Subunit, Bovine Brain

371768
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      Overview

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      Pricing & Availability

      Catalog NumberAvailability Packaging Qty/Pack Price Quantity
      371768-1250NG
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          Plastic ampoule 1250 ng
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          Description
          OverviewNative Gβγ-subunit purified from bovine brain. Provides sufficient G-protein βγ-subunit for six standard curves. The βγ-subunit is biologically active and can be used in reconstitution experiments. Useful for quantitation of β-subunit present in any particular cellular compartment.
          Catalogue Number371768
          Brand Family Calbiochem®
          References
          ReferencesBlank, J.L., et al. 1992. J. Biol. Chem. 267, 23069.
          Bourne, H.R., et al. 1992. Cold Spring Harbor Symp. Quant. Biol. 57, 145.
          Federman, A.D., et al. 1992. Nature 356, 159.
          Lustig, K.D., et al. 1993. J. Biol. Chem. 268, 13900.
          Pitcher, J.A., et al. 1992. Science 257, 1264.
          Tang, W.J., and Gilman, A.G. 1992. Cell 70, 869.
          Product Information
          FormLiquid
          FormulationIn 50 mM HEPES, 1 mM DTT, 1 mM EDTA, 0.1% LUBROL® Detergent, pH 7.6.
          PreservativeNone
          Applications
          ApplicationG-Protein, βγ-Subunit, Bovine Brain. Provided in amounts sufficient for six standard curves. It is biologically active and can be used in reconstitution experiments and for quantitation of β-subunit.
          Key Applications Immunoblotting (Western Blotting)
          Application NotesImmunoblotting (see comments)
          Application CommentsMETHOD OF ASSAY:

          1. The stock solution contains 1250 ng of βγ in 25 μl. Prepare working standards from this stock solution as follows: Add 100 μl of solubilization buffer [20 mM Tris, 1 mM EDTA, 1 mM DTT, 100 mM NaCl and 0.9% sodium cholate (pH 8.0)] and mix thoroughly. This provides a concentration of 10 ng/μl. Serially dilute with solubilization buffer to produce 5 ng/μl, 2.5 ng/μl, 1.25 ng/μl and 0.625 ng/μl. You should then have 62.5 μl of each of the standards except the 0.625 ng/μl which should be 125 μl. Complete preparation of the standards by adding 93.75 μl of sample buffer containing glycerol (25% w/v), tracking dye (0.05% w/v), SDS (5% w/v), glycine (3.6% w/v), and β-mercaptoethanol (10% v/v) to each standard except the 0.625 ng/μl, where you should add 187.5 μl to produce the desired concentration.
          A final standard containing the solubilization buffer and sample buffer in the ratio of 1:1.5 (10 μl solubilization buffer per 15 μl sample buffer) should also be prepared to give a zero standard. All standards should then be heated to 80°C for 4 min. The formulation described above provides 100, 50, 25, 12.5, and 6.25 ng of the βγ-subunit per 25 μl of volume. Alternative formulations are acceptable as long as these amounts of βγ-subunit are present for the preparation of a standard curve.
          2. The membrane or cellular organelle preparations should be solubilized (use the solubilization buffer described previously) on ice for 1 h. Thereafter, the preparation should be centrifuged to remove insoluble material; the amount of protein should be determined in the supernatant by an appropriate method. The solubilized protein should be diluted with solubilization buffer to obtain the desired amount of protein to be loaded in a volume of 10 μl. For each 10 μl of solubilized protein add 15 μl of the sample buffer. The preparations should be heated for 4 min at 80°C.
          3. Twenty-five (25) μl of each standard or sample should be loaded in each lane of a SDS-PAGE gel for electrophoresis by standard method. The resolved proteins should be transferred onto nitrocellulose or PVDF membranes by electrophoresis by standard methods. After blocking for 1 h with a standard blocking buffer [5% dried skim milk in buffer (pH 8.0) containing 50 mM Tris, 2 mM CaCl2, 80 mM NaCl, 0.02% sodium azide, and 0.2% Nonidet P-40] the blot should be probed with the provided primary antibody diluted by a factor of 1:1000 in the blocking buffer for at least 1.5 h. After washing, the bands can be visualized by the method of your choice. Use iodinated goat anti-rabbit IgG when your intent is to construct a standard curve using densitometric readings from the blot.
          Biological Information
          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 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).
          Canadian export regulations Due to the country and/or U.S. state of origin of the animal material used in this product, this product may not be exported to Canada.
          Packaging Information
          Transport Information
          Supplemental Information
          Specifications

          Documentation

          SDS

          Title

          Safety Data Sheet (SDS) 

          Certificates of Analysis

          TitleLot Number
          371768

          References

          Reference overview
          Blank, J.L., et al. 1992. J. Biol. Chem. 267, 23069.
          Bourne, H.R., et al. 1992. Cold Spring Harbor Symp. Quant. Biol. 57, 145.
          Federman, A.D., et al. 1992. Nature 356, 159.
          Lustig, K.D., et al. 1993. J. Biol. Chem. 268, 13900.
          Pitcher, J.A., et al. 1992. Science 257, 1264.
          Tang, W.J., and Gilman, A.G. 1992. Cell 70, 869.
          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.

          Revision13-May-2008 JSW
          ApplicationImmunoblotting (see comments)
          DescriptionMany membrane receptors for neurotransmitters and hormones are coupled to intracellular effectors and ion channels by heterotrimeric G-proteins. Receptor activation by ligand causes exchange, in a catalytic fashion, of GDP by GTP on the β-subunits of heterotrimeric G-proteins, followed by the dissociation of the β-subunit from the βγ-subunits. The latter reaction leads to regulation of a variety of effector systems. Recent evidence suggests that in addition to the β-subunit the βγ-subunits may also play a role in effector system regulation. The βγ-subunit has been implicated in the regulation of K+ channels, phospholipase C and A2, and type II adenylylcyclase. A recent study demonstrated a role for βγ-subunits in targeting the β-adrenergic receptor kinase to membrane-bound receptors as well. This product is useful for quantification of β-subunit present in any particular cellular compartment and to study the role of G-protein in intracellular signaling. The purified standard is suitable for positive control or can be used to construct a standard curve using Anti-G-Protein, β-Subunit, Internal (127-139) RAbbit pAb (Cat. No. 371738).
          BackgroundUseful for quantification of β-subunit present in any particular cellular compartment and to study the role of G-protein in intracellular signaling. The purified standard is suitable for positive control or can be used to construct a standard curve using Anti-G-Protein, β-Subunit, Internal (127-139) (Cat. No. 371738). Many membrane receptors for neurotransmitters and hormones are coupled to intracellular effectors and ion channels by heterotrimeric G-proteins. Receptor activation by ligand causes exchange, in a catalytic fashion, of GDP by GTP on the β-subunits of heterotrimeric G-proteins, followed by the dissociation of the β-subunit from the βγ-subunits. The latter reaction leads to regulation of a variety of effector systems. Recent evidence suggests that in addition to the β-subunit the βγ-subunits may also play a role in effector system regulation. The βγ-subunit has been implicated in the regulation of K+ channels, phospholipase C and A2, and type II adenylylcyclase. A recent study demonstrated a role for βγ-subunits in targeting the β-adrenergic receptor kinase to membrane-bound receptors as well.
          FormLiquid
          FormulationIn 50 mM HEPES, 1 mM DTT, 1 mM EDTA, 0.1% LUBROL® Detergent, pH 7.6.
          Concentration Label Please refer to vial label for lot-specific concentration
          Recommended reaction conditions
          1. The stock solution contains 1250 ng of βγ in 25 μl. Prepare working standards from this stock solution as follows: Add 100 μl of solubilization buffer [20 mM Tris, 1 mM EDTA, 1 mM DTT, 100 mM NaCl and 0.9% sodium cholate (pH 8.0)] and mix thoroughly. This provides a concentration of 10 ng/μl. Serially dilute with solubilization buffer to produce 5 ng/μl, 2.5 ng/μl, 1.25 ng/μl and 0.625 ng/μl. You should then have 62.5 μl of each of the standards except the 0.625 ng/μl which should be 125 μl. Complete preparation of the standards by adding 93.75 μl of sample buffer containing glycerol (25% w/v), tracking dye (0.05% w/v), SDS (5% w/v), glycine (3.6% w/v), and β-mercaptoethanol (10% v/v) to each standard except the 0.625 ng/μl, where you should add 187.5 μl to produce the desired concentration. A final standard containing the solubilization buffer and sample buffer in the ratio of 1:1.5 (10 μl solubilization buffer per 15 μl sample buffer) should also be prepared to give a zero standard. All standards should then be heated to 80°C for 4 min. The formulation described above provides 100, 50, 25, 12.5, and 6.25 ng of the βγ-subunit per 25 μl of volume. Alternative formulations are acceptable as long as these amounts of βγ-subunit are present for the preparation of a standard curve. 2. The membrane or cellular organelle preparations should be solubilized (use the solubilization buffer described previously) on ice for 1 h. Thereafter, the preparation should be centrifuged to remove insoluble material; the amount of protein should be determined in the supernatant by an appropriate method. The solubilized protein should be diluted with solubilization buffer to obtain the desired amount of protein to be loaded in a volume of 10 μl. For each 10 μl of solubilized protein add 15 μl of the sample buffer. The preparations should be heated for 4 min at 80°C. 3. Twenty-five (25) μl of each standard or sample should be loaded in each lane of a SDS-PAGE gel for electrophoresis by standard method. The resolved proteins should be transferred onto nitrocellulose or PVDF membranes by electrophoresis by standard methods. After blocking for 1 h with a standard blocking buffer [5% dried skim milk in buffer (pH 8.0) containing 50 mM Tris, 2 mM CaCl2, 80 mM NaCl, 0.02% sodium azide, and 0.2% Nonidet P-40] the blot should be probed with the provided primary antibody diluted by a factor of 1:1000 in the blocking buffer for at least 1.5 h. After washing, the bands can be visualized by the method of your choice. Use iodinated goat anti-rabbit IgG when your intent is to construct a standard curve using densitometric readings from the blot.
          PreservativeNone
          CommentsMETHOD OF ASSAY:

          1. The stock solution contains 1250 ng of βγ in 25 μl. Prepare working standards from this stock solution as follows: Add 100 μl of solubilization buffer [20 mM Tris, 1 mM EDTA, 1 mM DTT, 100 mM NaCl and 0.9% sodium cholate (pH 8.0)] and mix thoroughly. This provides a concentration of 10 ng/μl. Serially dilute with solubilization buffer to produce 5 ng/μl, 2.5 ng/μl, 1.25 ng/μl and 0.625 ng/μl. You should then have 62.5 μl of each of the standards except the 0.625 ng/μl which should be 125 μl. Complete preparation of the standards by adding 93.75 μl of sample buffer containing glycerol (25% w/v), tracking dye (0.05% w/v), SDS (5% w/v), glycine (3.6% w/v), and β-mercaptoethanol (10% v/v) to each standard except the 0.625 ng/μl, where you should add 187.5 μl to produce the desired concentration.
          A final standard containing the solubilization buffer and sample buffer in the ratio of 1:1.5 (10 μl solubilization buffer per 15 μl sample buffer) should also be prepared to give a zero standard. All standards should then be heated to 80°C for 4 min. The formulation described above provides 100, 50, 25, 12.5, and 6.25 ng of the βγ-subunit per 25 μl of volume. Alternative formulations are acceptable as long as these amounts of βγ-subunit are present for the preparation of a standard curve.
          2. The membrane or cellular organelle preparations should be solubilized (use the solubilization buffer described previously) on ice for 1 h. Thereafter, the preparation should be centrifuged to remove insoluble material; the amount of protein should be determined in the supernatant by an appropriate method. The solubilized protein should be diluted with solubilization buffer to obtain the desired amount of protein to be loaded in a volume of 10 μl. For each 10 μl of solubilized protein add 15 μl of the sample buffer. The preparations should be heated for 4 min at 80°C.
          3. Twenty-five (25) μl of each standard or sample should be loaded in each lane of a SDS-PAGE gel for electrophoresis by standard method. The resolved proteins should be transferred onto nitrocellulose or PVDF membranes by electrophoresis by standard methods. After blocking for 1 h with a standard blocking buffer [5% dried skim milk in buffer (pH 8.0) containing 50 mM Tris, 2 mM CaCl2, 80 mM NaCl, 0.02% sodium azide, and 0.2% Nonidet P-40] the blot should be probed with the provided primary antibody diluted by a factor of 1:1000 in the blocking buffer for at least 1.5 h. After washing, the bands can be visualized by the method of your choice. Use iodinated goat anti-rabbit IgG when your intent is to construct a standard curve using densitometric readings from the blot.
          Storage ≤ -70°C
          Avoid freeze/thaw
          Do Not Freeze Ok to freeze
          Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
          Toxicity Standard Handling
          ReferencesBlank, J.L., et al. 1992. J. Biol. Chem. 267, 23069.
          Bourne, H.R., et al. 1992. Cold Spring Harbor Symp. Quant. Biol. 57, 145.
          Federman, A.D., et al. 1992. Nature 356, 159.
          Lustig, K.D., et al. 1993. J. Biol. Chem. 268, 13900.
          Pitcher, J.A., et al. 1992. Science 257, 1264.
          Tang, W.J., and Gilman, A.G. 1992. Cell 70, 869.