Protein G Agarose

Description:
Protein G Agarose is useful tools for purification antibodies and immunoprecipitation(IP). Samples containing IgG are incubated with Protein G Agarose in a buffer that facilitates binding. After non-IgG and nonantigen components of the sample are washed from the resin, and the bound IgG and antigen may be recovered by elution.

Ligand: Recombinant Protein G
Bead size: ~ 40 µm
Binding capacity: High binding capacity, 10 µL Protein G Agarose bind about 50 µg of Human IgG.
Storage: Shipped at ambient temperature. Do not freeze.
Storage Buffer: 50 % slurry in PBS containing 20 % Ethanol

Background:
Protein G is a bacterial cell wall protein isolated from group G Streptococci. DNA sequencing of native Protein G identifies two IgG-binding domains and sites for albumin and cell surface binding. The albumin and cell surface binding domains have been eliminated from Recombinant Protein G to reduce nonspecific binding and, therefore, can be used to separate IgG from crude samples. Optimal binding occurs at pH 5, although binding is also good at pH 7.0-7.2. Because Protein G has greater affinity than Protein A for most mammalian IgGs, it may be used for the purification of mammalian IgGs that do not bind well to Protein A. Protein G binds with significantly greater capacity than Protein A to several IgG subclasses such as human IgG3, mouse IgG1 and rat IgG2a. However, Protein G does not bind to human IgM, IgD and IgA.

Immunoprecipitation (IP)/Co-IP
Protein Purification
Mass spectrometry (MS)
Enzyme activity measurements

Immunoprecipitation protocol

Mammalian cell lysis

Note: Harvesting of cells and cell lysis should be performed with ice-cold buffers. We strongly recommend to add protease inhibitors to the Lysis buffer to prevent degradation of your target protein and its binding partners.

For one immunoprecipitation reaction, we recommend using ~10⁶- 10⁷ cells.

1. Choice of lysis buffer:

* For cytoplasmic proteins, resuspend the cell pellet in 200 µL ice-cold Lysis buffer by pipetting up and down. Supplement Lysis buffer with protease inhibitor cocktail and 1 mM PMSF (not included).

* For nuclear/chromatin proteins, resuspend cell pellet in 200 µL ice-cold RIPA buffer supplemented with DNaseI (f.c. 75-150 Kunitz U/mL), MgCl2 (f.c. 2.5 mM), protease inhibitor cocktail and PMSF(f.c. 1 mM)(not included)

2. Place the tube on ice for 30 min and extensively pipette the suspension every 10 min.

3. Centrifuge cell lysate at 17,000x g for 10 min at +4°C. Transfer cleared lysate (supernatant) to a pre cooled tube and add 300 µL Dilution buffer supplemented with 1 mM PMSF and protease inhibitor cocktail (not included). If required, save 50 µL of diluted lysate for further analysis (input fraction).

Primary antibody binding

1.take supernant of the cell lysis into another tube and then add primary antibody (2ug-4ug/sample).

2.rotate at +4°C overnight.

Bead equilibration

1. Resuspend the beads by gently pipetting up and down or by inverting the tube. Do not vortex the beads!

2. Transfer 25 µL of bead slurry into a 1.5 mL reaction tube.

3. Add 500 µL ice-cold Dilution buffer.

4. Sediment the beads by centrifugation at 2,500x g for 5 min at +4°C.

5. Discard the supernatant.

Protein binding

1. Add diluted lysate to the equilibrated beads.

2. Rotate end-over-end for 1 hour at +4°C.

Washing

1. Sediment the beads by centrifugation at 2,500x g for 5 min at +4°C.

2. If required, save 50 µL of supernatant for further analysis(flow-through/non-bound fraction).

3. Discard remaining supernatant.

4. Resuspend beads in 500 µL Wash buffer.

5. Sediment the beads by centrifugation at 2,500x g for 5 min at +4°C. Discard the remaining supernatant.

6. Repeat this step at least twice.

7. During the last washing step, transfer the beads to a new tube.

Optional: To increase stringency of the Wash buffer, test various salt concentrations e.g. 150 mM - 500 mM,and/or add a non-ionic detergent e.g. Triton™ X-100.

Elution with 2x SDS-sample buffer

1. Remove the remaining supernatant.

2. Resuspend beads in 80 µL 2x SDS-sample buffer.

3. Boil beads for 5 min at +95°C to dissociate immunocomplexes from beads.

4. Sediment the beads by centrifugation at 2,500x g for 2 min at +4°C.

5. Analyze the supernatant in SDS-PAGE.

Elution with Glycine-elution buffer

1. Remove the remaining supernatant.

2. Add 50–100 µL Glycine-elution buffer and constantly pipette up and down for 30 - 60 sec at +4°C.

3. Sediment the beads by centrifugation at 2,500x g for 5 min at +4°C.

4. Transfer the supernatant to a new tube.

5. Immediately neutralize the eluate fraction with Neutralization buffer.

6. Repeat this step at least once to increase elution efficiency .