Promega G7471 Magne Protein G Beads Owner’s Manual

Promega G7471 Magne Protein G Beads

Specifications

Product Usage Instructions

1. Selection of Beads
   Choose between MagneTM Protein A Beads or MagneTM Protein G Beads based on the binding affinities of Protein A or Protein G for specific antibodies as outlined in Table 2.
   Product Components and StorageDo not reuse the beads provided. Refer to the product packaging  for specific storage conditions.

2. Preparation Steps
   Ensure that the beads remain in suspension during binding and wash steps for optimal antibody yield and purity. Use a tube shaker or end-over-end mixer for this purpose.

3. Concentration of Cell Culture

4. Medium Prior to antibody purification, concentrate cell culture medium  using a low-molecular-weight-cutoff filter to reduce sample volume and increase antibody concentration. Scale all volumes proportionally to minimize sample dilution.

5. Buffer Compatibility
   Use bind/wash buffers like sodium acetate buffer, phosphate-buffered saline, or Tris-buffered saline with the beads. For elution, use glycine-HCl buffer, and for neutralization, use Tris buffer.

6. Protocol Optimization
   Note that different antibody isotypes or antibodies from various species may require protocol optimization for maximal recovery and compatibility with downstream applications.

FAQ
Q: Can I reuse the MagneTM Protein A or MagneTM Protein G Beads?
A: No, it is not recommended to reuse the beads for antibody purification.

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

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 encapsulated by macroporous cellulose, resulting in low non-specific binding. The beads also have excellent magnetic properties that allow rapid and efficient capture using a variety of magnetic stands (see Section 7).
Both beads use a novel attachment chemistry based on the HaloTag® technology to immobilize Protein A or Protein G. The HaloTag® protein is a multi- functional protein tag that forms a covalent bond with its ligand and is used for protein expression and purification, cell imaging, protein immobilization and protein-interaction analysis (see www.promega.com/halotag/). The Magne™ Protein A Beads and Magne™ Protein G Beads use recombinant Protein A from Staphylococcus and Protein G from Streptococci, respectively, fused with the HaloTag® protein. The Protein
A-HaloTag® (M.Wt 71kDa) or Protein G-HaloTag® (M.Wt 58kDa) fusion protein is covalently attached in an oriented fashion to a magnetic cellulose bead that is activated using a HaloTag® ligand. The bead characteristics are shown in Table 1.

Table 1. Characteristics of the Magne™ Protein A and Magne™ Protein G Beads.

• 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.
• Magne™ Protein A Beads and Magne™ Protein G Beads are not recommended for use in immunoprecipitation (IP) or co-IP applications.
• Magne™ Protein G Beads are optimized for immunoglobulin purification. Do not use the Magne™ HaloTag® Beads  (Cat.# G7281) or HaloLink™ Resin (Cat.# G1912) to bind the Protein G-HaloTag® Fusion Protein (Cat.# G7291), as these products are not optimized for immunoglobulin purification.
• Special Acknowledgement: The Magne™ Protein G Beads, Protein G HaloTag® Fusion Protein and Magne™ Protein A Beads were developed in collaboration with Kazusa DNA Research Institute (KDRI). We would like to thank KDRI for their technical guidance and early testing.

Table 2. Binding Affinity of Protein A and Protein G for Different Antibodies and Isotypes

Product Components and Storage Conditions

Storage Conditions: Store the Magne™ Protein A and Magne™ Protein G Beads at +2°C to +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.

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) and Magne™ Protein G Beads (G) as described in this Technical Manual. Samples were analyzed by adding 1µl of starting material (SM) or 5µl of purified sample to SDS buffer, and heating at 80°C for ten minutes before loading onto a 4–20% Tris-glycine gel. The gel was stained with SimplyBlue® Safe Stain.

Before You Begin

1. 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.

2. 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.

3. Sample incubation times may need to be optimized. Binding may be performed at 4°C; however, longer incubation times may be necessary for efficient antibody capture.

4. Biological samples may be cleared by filtration through a 0.22µm filter or by a 15-minute centrifugation at
   14,000 × g to remove aggregates and insoluble proteins prior to antibody purification.

5. Prior to antibody purification, cell culture medium can be concentrated using a low-molecular-weight-cutoff filter (e.g., molecular weight cutoff of 3,500 Daltons) to reduce the starting sample volume and increase the antibody concentration. Scale the sample, bind/wash buffer and Magne™ Protein A Bead or Magne™ Protein G Bead volumes proportionally. To minimize sample dilution, a 10X bind/wash buffer may be added to the sample to a final concentration of 1X.

6. Magne™ Protein A Beads and Magne™ Protein G Beads are compatible with several bind/wash buffers, including 25mM sodium acetate buffer (pH 6.0), phosphate-buffered saline (pH 7.4) and Tris-buffered saline (pH 7.5). We recommend an elution buffer of 100mM glycine-HCl (pH 2.7) and a neutralization buffer of 2M Tris buffer (pH 7.5). Note: Purification of different antibody isotypes or antibodies from different species may require protocol
   ! optimization for maximal recovery and compatibility with downstream applications.

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.

Figure 2. Schematic of antibody purification using Magne™ Protein A Beads or Magne™ Protein G Beads. Inset: Magne™ Protein A Beads captured on a magnet.

Table 3. Guidelines for Antibody Purification.

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 aliqotting beads.
2.  Add 50µl of bead slurry to a 1.5ml microcentrifuge tube. Place in the magnetic stand for 10 seconds.
3.  Remove and discard the storage buffer.
4. Add 500µl of bind/wash buffer. Mix and place in the magnetic stand for 10 seconds. Remove and discard the bind/wash buffer.
5. Combine 50µl of bind/wash buffer and 50µl of sample, then add to the equilibrated beads.
6. Mix sample for 30–60 minutes at room temperature. Make sure the beads remain in suspension by using a tube shaker or end-over-end mixer.
7. Place tube in the magnetic stand for 10 seconds. Remove the supernatant, and save for analysis if desired.
8. Wash beads by adding 500µl of bind/wash buffer and mix for 5 minutes. Place in the magnetic stand for
9. seconds. Remove and discard bind/wash buffer.
10. Repeat Step 8 for a total of two washes.
11. Wash beads by adding 200µl of bind/wash buffer. Mix and place in the magnetic stand for 10 seconds. Remove and discard all bind/wash buffer.
12. add 50µl of elution buffer [100mM glycine-HCl (pH 2.7)] to the beads.
13. Mix for 5 minutes at room temperature.
14. Place tube in the magnetic stand for 10 seconds. Remove eluted sample, and transfer to a new microcentrifuge tube containing 10µl of neutralization buffer [2M Tris buffer (pH 7.5)]. This is the first elution.
15. Repeat elution Steps 11–13. Eluted samples can be combined.
16. Quantitate the amount of antibody recovered by measuring absorbance at 280nm or using a protein quantitation method such as a Bradford assay. ELISA may be a better quantitative method for samples with low antibody concentration. Check the purity of the antibody using SDS polyacrylamide gel electrophoresis.

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

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 final volume to 10ml with deionized water.
• Bind/Wash Buffers
• PBS (pH 7.4) 137mM NaCl
• 2.68mM KCl 1.47mM KH2PO4
• 8.1mM Na2HPO4
• 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) 150mM NaCl

Related Products

Summary of Changes

The following changes were made to the 1/24 revision of this document:

1. Updated patent statements.
2. Changed font and cover image.
3. Made minor text edits.

• (a)U.S. Pat. No. 10,604,745 and other patents pending.
• © 2012–2024 Promega Corporation. All Rights Reserved.
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• Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.
• All prices and specifications are subject to change without prior notice.
• Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.

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TM371 · Revised 1/24

References

• Promega Corporation
• Protocols

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