Dynabeads Protein G: Product Contents Required Materials Protocol
Dynabeads Protein G: Product Contents Required Materials Protocol
Dynabeads Protein G
Catalog nos. 10003D, 10004D, 10009D
Store at 2C to 8C
Product Contents
Cat. no.
Volume
10003D
1 mL
10004D
5 mL
10009D
50 mL
Product Description
Dynabeads Protein G are designed
for immunoprecipitation of proteins,
protein complexes, protein-nucleic acid
complexes, and other antigens.
Antibody (Ab) is added to the
Dynabeads Protein G. During a
short incubation, the Ab binds to the
Dynabeads via their Fc-region. The
tube is then placed on a magnet, where
the beads migrate to the side of the
tube facing the magnet and allow for
easy removal of the supernatant. The
bead-bound Ab may now be used for
immunoprecipitation. Bound material
is easily collected utilizing the unique
magnetic properties of the Dynabeads.
Required Materials
Protocol
Cell Lysis
Cells may be lysed using any standard cell lysis protocol compatible with your
starting material. We recommend the use of Cell Extraction Buffer or NP40 Cell Lysis
Buffer. For protocols and additional information about cell lysis, see
www.lifetechnologies.com/immunoprecipitation.
Prepare Dynabeads
1. Resuspend Dynabeads in the vial (vortex >30 sec or tilt and rotate 5 min).
2. Transfer 50 L (1.5 mg) Dynabeads to a tube.
3. Place the tube on the magnet to separate the beads from the solution, and remove
the supernatant.
4. Remove the tube from the magnet.
5. Proceed directly to Binding of Antibody.
Bind Antibody
General Guidelines
1. Add your antibody (Ab) (typically 110 g) diluted in 200 L PBS with
Tween-20, to the Dynabeads from step 4 in "Prepare Dynabeads". The optimal
amount of Ab needed depends upon the individual Ab used.
Figure 1: Principle of immunoprecipitation of
antigen using Dynabeads Protein G.
For research use only. Not for human or animal therapeutic or diagnostic use.
Crosslinking
To avoid co-elution of your antibody, crosslink your antibody to the Dynabeads
before continuing with immunoprecipitation. Use the crosslinking reagent BS3. For
further information and procedure, visit www.lifetechnologies.com/crosslinking.
4. Place the tube on the magnet. Transfer the supernatant to a clean tube for further
analysis, if desired.
5. Wash the Dynabeads-Ab-Ag complex 3 times using 200 L Washing Buffer for
each wash. Separate on the magnet between each wash, remove supernatant and
resuspend by gentle pipetting.
6. Resuspend the Dynabeads-Ab-Ag complex in 100 L Washing Buffer and
transfer the bead suspension to a clean tube. This is recommended to avoid coelution of proteins bound to the tube wall.
Human IgG1,2,4
+++
Human IgD
Human IgA, E, M
Human IgG3
+++
Mouse IgG1
+++
+++
Mouse IgM
Rat IgG1
Rat IgG2a
+++
Rat IgG2b
Rat IgG2c
Bovine IgG1
+++
Bovine IgG2
+++
Chicken IgY
Dog IgG
5. Place the tube on the magnet and load the supernatant/sample onto a gel.
Goat IgG1
+++
Goat IgG2
+++
Hamster
NA
Horse IgG
+++
Monkey IgG
+++
Porcine IgG
+++
Rabbit IgG
+++
3. Incubate with rotation for 2 min at room temperature to dissociate the complex.
Sheep IgG1
+++
4. Place the tube on the magnet and transfer the supernatant containing eluted Ab
and Ag to a clean tube. If the eluted protein is to be used for functional assays or
stored, the pH of the eluate can be adjusted by adding 1 M Tris, pH 7.5.
Sheep IgG2
+++
EluteTarget Antigen
A. Denaturing elution
1. Place the tube (from step 7 in "Immunoprecipitation of Target Antigen") on the
magnet and remove the supernatant.
2. Add 20 L Elution Buffer, and 10 L premixed NuPAGE LDS Sample Buffer and
NuPAGE Sample Reducing Agent (mixed as per manufacturers instructions).
B. Non-denaturing elution
1. Place the tube (from step 7 in "Immunoprecipitation of Target Antigen") on the
magnet and remove the supernatant.
2. Add 20 L Elution Buffer and gently pipette to resuspend the Dynabeads-Ab-Ag
complex. Avoid foaming.
Description of Materials
This product contains Dynabeads Protein G for immunoprecipitation. Dynabeads
Protein G are uniform, 2.8 m, superparamagnetic beads with recombinant Protein G
(approximately 45 kDa) covalently coupled to the surface.
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