Vanderbilt Antibody
and Protein Resource
Vanderbilt Antibody and Enzyme Repository
Available via the Molecular Biology Core (http://thecore.vanderbilt.edu/)
Anti-Myc 9E10 on Magnetic/Sepharose Beads
Antibody Type: Mouse Monoclonal
Isotype: IgG1
Clone: 9E10
Immunogen: synthetic peptide derived from amino acids 408-439 of c-Myc protein
Lot: 131024
Lot Binding Capacity: 1mg/ml of slurry
Formulation: 2mgs of settled beads in 2mLs PBS + 0.5% Sodium Azide
APPLICATIONS
Assay
Recommended
Concentration
ELISA
No
WB
No
IP
IF
IHC
1ml bead
No
No
slurry binds
~1mg target
protein
Note: Optimal dilutions should be established by the end user. These concentrations are
intended as a guide.
LIGAND COUPLING STABILITY AND TARGET ELUTION EFFICIENCY
Elution Conditions
Reducing buffer
+ Boiling
Non-reducing
buffer+ Boiling
Low pH:
Glycine pH 2.0
High salt:
3.6 M MgCl
Magnetic Beads
Bound Ligand
Leakage
1-3%
Target Elution
Efficiency (i.e. GST)
100%
Sepharose Beads
Bound Ligand
Leakage
0-2%
Target Elution
Efficiency (i.e. GST)
100%
1-3%
≈50% - 75%
0-2%
≈50% - 75%
1-2%
≈15% - 35%
1-2%
≈15% - 35%
BDL
≈5% - 20%
BDL
≈5% - 20%
Bound ligand leakage = the estimated percentage of GBP protein that will be present in the
elute using the indicated elution conditions.
Target Elution Efficiency = the estimated percentage of bound GFP target protein that was
eluted using the indicated elution conditions. GFP eluted using Reducing buffer + boiling was
considered 100%, all others were quantified relative to this condition.
BDL= Below Detection Limits (0.001%)
www.vanderbilt.edu/vapr
Phone: 936-3092
Room: 892 PRB
SUGGESTED PROTOCOL
Note: The following is a standard Immunoprecipitation protocol and may need to be adjusted
based on the user’s experimental conditions. The theoretical binding capacity is 1mg/ml of
bead slurry. However, in practice we have generally found the binding capacity to be
approximately 0.5-0.8mg/ml of bead slurry.
Protocol
1.
2.
3.
4.
5.
Thoroughly mix beads and storage buffer
Remove desired amount of bead slurry
Place on magnet for 2 minutes
Remove storage buffer
Wash with 300-500uL of 1X PBS three times; mixing/vortexing, spinning down, and
placing on magnet each time to remove the PBS
6. Add in target containing lysate/buffer/etc.
7. Rotate end-over-end for 2 hours at room temperature or overnight at 4°C
8. Spin down sample and place on magnet
9. Remove unbound protein in solution for use as flow-through sample
10. Wash beads three times with PBS, removing wash buffer each time (Step 5)
11. If necessary retain washes to examine on a SDS-PAGE gel or Western Blot.
12. Elution conditions will vary across experiments. Four different elution conditions
have been tested in VAPR and their data is shown above on page 1.
13. If using Protein Gel Loading Buffer as the elution strategy add desired volume of
Sample Loading Buffer and proceed with boiling and/or gel loading.
14. If eluting via Glycine or High Salt,
a. Add 3 volumes of elution buffer and incubate for 3 to 5 minutes.
b. Spin down and place on magnet.
c. Remove eluted sample. Please note that Glycine eluted samples need to
have their pH neutralized and High Salt eluted samples will need to be buffer
exchanged before running on SDS-PAGE.
STORAGE AND UTILIZATION
Antibodies bound to solid matrices are provided in PBS with 0.05% Sodium azide. Stored at
4˚C, the solution will be stable for 6 months or longer.
All VAPR products are quality tested and fully guaranteed.
If you have any issues please contact us.
www.vanderbilt.edu/vapr
Phone: 936-3092
Room: 892 PRB