Magne Protein G Beads and Magne Protein A Beads
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TECHNICAL MANUAL Magne™ Protein A Beads and Magne™ Protein G Beads for Antibody Purification Instructions for Use of Products G7471, G7472, G7473, G8781, G8782 and G8783

Magne™ Protein A Beads and Magne™ Protein G Beads for Antibody Purification All technical literature is available at: www.promega.com/protocols/ Visit the web site to verify that you are using the most current version of this Technical Manual. E-mail Promega Technical Services if you have questions on use of this system: techserv@promega.com 1. Description Magne™ Protein A Beads and Magne™ Protein G Beads(a) are magnetic affinity beads with high specificity and high capacity for binding immunoglobulins from cell culture, ascites and serum samples. These magnetic beads are composed of iron...

Composition Magnetic bead based on macroporous cellulose Chemistry Oriented and covalent attachment of Protein A or Protein G using HaloTag® technology Particle Size 30-80pm. Antibody-Binding Capacity >18mg of human IgG/1ml of settled beads Formulation 20% slurry in 20% ethanol The choice between selecting Magne™ Protein A Beads or Magne™ Protein G Beads depends on the difference in the binding affinities of Protein A or Protein G for antibodies from different species and for different antibody isotypes. See Table 2. • High purity and recovery of antibodies • No...

Table 2. Binding Affinity of Protein A and Protein G for Different Antibodies and Isotypes Species/Subclass Protein A Protein G Rabbit Cow Goat Chicken Human IgG Human IgM Human IgD Human IgA Key: Strong (+++), Medium (++), Weak (+). Adapted from Akerstrom, B. et al. (1985) J. Immunol. 135(4), 2589-92. Promega Corporation ■ 2800 Woods Hollow Road ■ Madison, WI 53711-5399 USA ■ Toll Free in USA 800-356-9526 ■ 608-274-4330 ■ Fax 608-277-2516 3

Product Components and Storage Conditions Magne™ Protein A Beads Magne™ Protein G Beads Storage Conditions: Store the Magne™ Protein A and Magne™ Protein G Beads at 2–10°C. Do not freeze. Do not allow beads to dry during storage or use. Do not reuse the Magne™ Protein A or Magne™ Protein G Beads. Cell Media kDa Albumin IgG heavy chain (55kDa) Figure 1. Antibody purified from various sample types using the Magne™ Protein A and Magne™ Protein G Beads. Antibody was purified from 50µl of cell culture media (mouse IgG1), mouse ascites (IgG2a) and goat serum using 50µl Magne™ Protein A Beads (A)...

Antibody purification using Magne™ Protein A Beads or Magne™ Protein G Beads requires a magnetic stand. The beads can be used with a variety of magnetic stands available from Promega (see Section 7) to purify antibody from 1–96 samples in parallel, with sample volumes ranging from 20µl to 30ml. Be sure that the Magne™ Protein A Beads and Magne™ Protein G Beads remain in suspension during binding and wash steps for maximal antibody yield and purity. We recommend using a tube shaker or end-over-end mixer. Sample incubation times may need to be optimized. Binding may be performed at 4°C;...

Antibody Purification Protocol Figure 2 shows a schematic of the antibody purification protocol using Magne™ Protein A Beads or Magne™ Protein G Beads. The volume of beads, bind/wash buffer and elution buffer can be scaled proportionally to accommodate different sample volumes and sample types. See Table 3 for guidelines on processing different sample sizes. Magne™ Protein G Beads or Magne™ Protein A Beads Add biological sample containing antibodies. Incubate for 30–60 minutes. Antibodies bound to beads Wash to remove nonspecific proteins. MagneSphere® Technology Magnetic Separation Stand....

Table 3. Guidelines for Antibody Purification.Sample Type Serum, Ascites, Cell Culture Cell Culture Cell Culture Media Media Media Cell Culture Media Serum, Ascites, Cell Culture Media Format 1.5ml tubes 1.5ml tubes 15ml conical 50ml conical 96-well plate Equilibrate Beads Magnetic Stand (See Section 7) Amount of Bind/Wash Buffer to Dilute Sample Bind Time Wash with Bind/Wash Buffer 50pl of 1X bind/ wash buffer Deep-Well PolyATtract® System 1000 MagnaBot® 96 Magnetic Separation Stand Magnetic Separation (Z5410) Device 1ml of 10X bind wash buffer 5ml of 10X bind wash buffer...

Antibody Purification Protocol (continued) This protocol is for manual antibody purification from 50µl of serum, ascites or cell culture media in a microcentrifuge tube format. See Table 3 for the volume of beads, bind/wash buffer, elution buffer and neutralization buffer to use for different sample sizes. Materials to Be Supplied by the User (Solution compositions are provided in Section 6.) • bind/wash buffer • elution buffer • neutralization buffer • magnetic stand • mixing platform 1. Gently vortex or invert the beads to obtain a uniform suspension. Keep the suspension uniform when...

5. Troubleshooting For questions not addressed here, please contact your local Promega Branch Office or Distributor. Contact information available at: www.promega.com E-mail: techserv@promega.com Promega Corporation ■ 2800 Woods Hollow Road ■ Madison, WI 53711-5399 USA ■ Toll Free in USA 800-356-9526 ■ 608-274-4330 ■ Fax 608-277-2516 9

25mM Sodium Acetate (pH 6) 0.17g sodium acetate Dissolve sodium acetate in 40ml of deionized water. Adjust to pH 6 with HCl, Bring the final volume to 50ml with deionized water. Tris-buffered Saline (pH 7.5) 100mM Tris buffer (pH 7.5) 6. Composition of Buffers and Solutions Elution Buffer (100mM glycine-HCl, pH 2.7) 0.375g glycine Dissolve in deionized water. Adjust pH to 2.7 with HCl. Bring final volume to 50ml with deionized water. Neutralization Buffer (2M Tris buffer, pH 7.5) 0.472g Trizma base 2.54g Trizma hydrochloride Dissolve in deionized water. Adjust to pH 7.5. Bring the...

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