IP10 Protein G Plus/Protein A-Agarose

IP10
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IP10-10ML
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      Plastic ampoule 10 ml
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      Description
      OverviewDesigned for immunoglobulin purification at low pressure. Product size refers to the volume of packed beads.
      Catalogue NumberIP10
      Brand Family Calbiochem®
      References
      Product Information
      FormLiquid slurry
      Formulation50% suspension in PBS.
      Preservative≤0.1% sodium azide
      Applications
      Data setup Error. Pagelet <collection_feature_application_id-AB PUR> not found.
      Application NotesAntibody Purification
      Application CommentsNote: the product size refers to the volume of packed beads. This product can be used directly with serum, plasma, tissue culture media, ascites, or other biological fluids, but if sufficient quantities of starting material are available we recommend an initial clean up step. The column life will be greatly extended if aggregated proteins and lipids are removed from the immunoglobulin in the clean up step. Use 5-10 ml of packed beads per ml serum.

      Recommended Protocol for IgG Purification

      Buffers

      All concentrations stated are for working solutions, not the 10X concentrates. Caution: sodium azide is poison.

      Binding/Washing Buffer: 100 mM sodium phosphate pH 7.0, 150 mM sodium chloride, 5 mM sodium EDTA, 0.01% sodium azide.
      Elution Buffer A (see Note section): 500 mM ammonium acetate pH 3.0, 0.01% sodium azide.
      Elution Buffer B: 10 mM glycine/HCl pH 3.0, and 0.01% sodium azide.
      Neutralization Buffer: 500 mM Tris Base, 0.01% sodium azide.
      Storage Buffer: 100 mM sodium phosphate, pH 7.0, 0.01% sodium azide.

      Protocol

      A. Clean Up and Concentration
      Ascites and serum should be clotted at room temperature, refrigerated at 4°C overnight (to allow the clot to shrink and lipids to separate), and centrifuged multiple times to remove all clotted protein and lipid. Remove the lipid from the top of the centrifuge tube with a glass rod or small wooden stick. Tissue culture media should be centrifuged or filtered to remove aggregates.
      IgG can be concentrated and partially purified by use of an ammonium sulfate precipitation step. Add ammonium sulfate to 50% saturation (313 g per L) with stirring and check the pH adjusting to 7.0 by addition of 1 M HCl or NaOH. Centrifuge to collect precipitated immunoglobulin, dissolve in binding buffer and dialyze against the same buffer.

      B. Purification
      1. Pack a column with the Agarose Conjugate.
      2. Wash with about 20 column volumes Washing/Binding Buffer until pH of eluate is 7.0.
      3. If IgG has not been previously dialyzed against binding buffers dilute or dialyze IgG-containing sample into the Washing/Binding Buffer (pH 6.5-7.5).
      4. Load sample onto column.
      5. Wash with Washing/Binding Buffer until the absorbance of the eluate at 280 nm approaches background level.
      6. Wash with Elution Buffer A to elute IgG, and collect fractions until A280 returns to background levels.
      7. Wash with Elution Buffer B, and collect fractions until A280 returns to background. Most IgG should elute with buffer A.
      8. Neutralize eluted IgG fractions by addition of an equal volume of neutralization buffer and check the pH with pH paper. For best results, neutralize eluate promptly.
      9. To re-use the column immediately, repeat procedure from Step 2.
      10. To prepare the column for storage, wash column with 5 column volumes of Elution Buffer B.
      11. To store column wash with 30 column volumes storage buffer; then seal column outlets and store in refrigerator.
      12. Quantitate the purified IgG using the formula:

      Absorbance at 280 nm/1.4 = Concentration (mg/ml).

      To make Elution Buffer A, start with acetic acid and adjust the pH to 3.0 with ammonium hydroxide.
      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 Blue Ice Only
      Toxicity Standard Handling
      Storage +2°C to +8°C
      Do not freeze Yes
      Packaging Information
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      Protein G Plus/Protein A-Agarose SDS

      Title

      Safety Data Sheet (SDS) 

      Protein G Plus/Protein A-Agarose Certificates of Analysis

      TitleLot Number
      IP10

      Citations

      Title
    • Ellen J. Tisdale and Cristina R. Artalejo. (2006) Src-dependent a protein kinase C(aPKC) λ tyrosine phosphorylation is required for aPKCl/λ association with Rab2 and glyceraldehyde-3-phosphate dehydrogenase on pre-golgi intermediates. Journal of Biological Chemistry 281, 8436-8442.
    • Douglas M. Molina, Seema Grewal and Lee Bardwell. (2005) Characterization of an ERK-binding domain in Microphthalmia-associated transcription factor and differential inhibition of ERK2-mediated substrate phosphorylation. Journal of Biological Chemistry 280, 42051-42060.
    • Ellen J. Tisdale, Carmen Kelly and Cristina R. Artalejo. (2004) Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to golgi transport exclusive of its glycolytic activity. Journal of Biological Chemistry 279, 54046-54052.
    • 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.

      Revision04-September-2007 RFH
      ApplicationAntibody Purification
      DescriptionA mixture of Protein G PLUS and Protein A covalently conjugated to agarose. Useful for purification of IgG from biological fluids.
      BackgroundThe protein is covalently coupled to the agarose support and does not leach. The protein A will bind IgG from biological fluids, but with different efficiencies, depending on the species. In general, Protein A works well for purification of IgG of large mammals, while Protein G PLUS and a mixture of Protein G PLUS with Protein A are best for rodent IgG purifications.
      FormLiquid slurry
      Formulation50% suspension in PBS.
      Preservative≤0.1% sodium azide
      CommentsNote: the product size refers to the volume of packed beads. This product can be used directly with serum, plasma, tissue culture media, ascites, or other biological fluids, but if sufficient quantities of starting material are available we recommend an initial clean up step. The column life will be greatly extended if aggregated proteins and lipids are removed from the immunoglobulin in the clean up step. Use 5-10 ml of packed beads per ml serum.

      Recommended Protocol for IgG Purification

      Buffers

      All concentrations stated are for working solutions, not the 10X concentrates. Caution: sodium azide is poison.

      Binding/Washing Buffer: 100 mM sodium phosphate pH 7.0, 150 mM sodium chloride, 5 mM sodium EDTA, 0.01% sodium azide.
      Elution Buffer A (see Note section): 500 mM ammonium acetate pH 3.0, 0.01% sodium azide.
      Elution Buffer B: 10 mM glycine/HCl pH 3.0, and 0.01% sodium azide.
      Neutralization Buffer: 500 mM Tris Base, 0.01% sodium azide.
      Storage Buffer: 100 mM sodium phosphate, pH 7.0, 0.01% sodium azide.

      Protocol

      A. Clean Up and Concentration
      Ascites and serum should be clotted at room temperature, refrigerated at 4°C overnight (to allow the clot to shrink and lipids to separate), and centrifuged multiple times to remove all clotted protein and lipid. Remove the lipid from the top of the centrifuge tube with a glass rod or small wooden stick. Tissue culture media should be centrifuged or filtered to remove aggregates.
      IgG can be concentrated and partially purified by use of an ammonium sulfate precipitation step. Add ammonium sulfate to 50% saturation (313 g per L) with stirring and check the pH adjusting to 7.0 by addition of 1 M HCl or NaOH. Centrifuge to collect precipitated immunoglobulin, dissolve in binding buffer and dialyze against the same buffer.

      B. Purification
      1. Pack a column with the Agarose Conjugate.
      2. Wash with about 20 column volumes Washing/Binding Buffer until pH of eluate is 7.0.
      3. If IgG has not been previously dialyzed against binding buffers dilute or dialyze IgG-containing sample into the Washing/Binding Buffer (pH 6.5-7.5).
      4. Load sample onto column.
      5. Wash with Washing/Binding Buffer until the absorbance of the eluate at 280 nm approaches background level.
      6. Wash with Elution Buffer A to elute IgG, and collect fractions until A280 returns to background levels.
      7. Wash with Elution Buffer B, and collect fractions until A280 returns to background. Most IgG should elute with buffer A.
      8. Neutralize eluted IgG fractions by addition of an equal volume of neutralization buffer and check the pH with pH paper. For best results, neutralize eluate promptly.
      9. To re-use the column immediately, repeat procedure from Step 2.
      10. To prepare the column for storage, wash column with 5 column volumes of Elution Buffer B.
      11. To store column wash with 30 column volumes storage buffer; then seal column outlets and store in refrigerator.
      12. Quantitate the purified IgG using the formula:

      Absorbance at 280 nm/1.4 = Concentration (mg/ml).

      To make Elution Buffer A, start with acetic acid and adjust the pH to 3.0 with ammonium hydroxide.
      Storage +2°C to +8°C
      Do Not Freeze Yes
      Toxicity Standard Handling
      Citation
    • Ellen J. Tisdale and Cristina R. Artalejo. (2006) Src-dependent a protein kinase C(aPKC) λ tyrosine phosphorylation is required for aPKCl/λ association with Rab2 and glyceraldehyde-3-phosphate dehydrogenase on pre-golgi intermediates. Journal of Biological Chemistry 281, 8436-8442.
    • Douglas M. Molina, Seema Grewal and Lee Bardwell. (2005) Characterization of an ERK-binding domain in Microphthalmia-associated transcription factor and differential inhibition of ERK2-mediated substrate phosphorylation. Journal of Biological Chemistry 280, 42051-42060.
    • Ellen J. Tisdale, Carmen Kelly and Cristina R. Artalejo. (2004) Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to golgi transport exclusive of its glycolytic activity. Journal of Biological Chemistry 279, 54046-54052.
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      Categories

      Life Science Research > Protein Detection and Quantification > Immunoassays > Immunoprecipitation (IP) > Agarose Beads for IP & Antibody Purification
      Life Science Research > Antibodies and Assays > Immunoassays > Immunoprecipitation (IP) > Agarose Beads for IP & Antibody Purification
      Life Science Research > Protein Sample Preparation > Protein Purification > Agarose Bead Affinity Purification > Agarose Beads for IP & Antibody Purification