Vanderbilt Antibody
and Protein Resource
Vanderbilt Antibody and Enzyme Repository
Available via the Molecular Biology Core (http://thecore.vanderbilt.edu/)
MYC Binding Sepharose Resin
Antibody Type: Recombinant antibody
Isotype: N/A
Immunogen: MYC 9E10
Lot: 130628
Lot Binding Capacity: 0.5mg/ml settled resin
Formulation: 50% slurry of resin in PBS + 0.5% Sodium Azide
APPLICATIONS
Assay
Recommended
Concentration
ELISA
No
WB
No
IP
IF
IHC
1ml settled
No
No
resin per
0.5mg 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. MYC)
100%
Sepharose Beads
Bound Ligand
Leakage
<0.05%
Target Elution
Efficiency (i.e. MYC)
100%
<1%
~75-95%
< 0.005%
~75-95%
<2%
~25-35%
BDL
~25-35%
BDL
~15-20%
BDL
~15-20%
Bound ligand leakage = the estimated percentage of MYC protein that will be present in the
elute using the indicated elution conditions.
Target Elution Efficiency = the estimated percentage of bound MYC target protein that was
eluted using the indicated elution conditions. MYC 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 4mg/ml of
settled resin. However, in practice we have generally found the binding capacity to be
approximately 0.5mg/ml of settled resin.
Protocol
1. Thoroughly mix beads and storage buffer
2. Remove desired amount of resin (mixed resin:buffer is a 50:50 slurry)
3. Centrifuge resin slurry at 1,000 x g for 2 minutes
4. Remove storage buffer
5. Wash with 5 volumes of PBS where the volume represents amount of settled resin
6. Centrifuge resin slurry at 1,000 x g for 2 minutes
7. Remove PBS
8. Add in MYC containing lysate/buffer/etc.
9. Rotate end-over-end for 1 hour at room temperature or 2 hours at 4°C
10. Centrifuge sample at 1,000 x g for 2 minutes
11. Remove unbound protein in solution for use as flow-through sample
12. Wash beads with 5 volumes of PBS (or appropriate wash buffer)
13. Centrifuge resin slurry at 1,000 x g for 2 minutes
14. Remove wash buffer
15. Repeat steps 12 through 14 two times. If necessary retain washes to examine on
SDS-PAGE.
16. Elution conditions will vary across experiments. Four different elution conditions
have been tested in VAPR and their data is shown above on page 1.
17. 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.
18. If eluting via Glycine or High Salt,
a. add 3 volumes of elution buffer and incubate for 3 to 5 minutes.
b. Centrifuge at 1,000 x g for 2 minutes
c. Remove eluted sample. Please note that Glycine eluted samples with need
to have their pH neutralized and High Salt eluted samples will need to be
buffer exchanged before running on SDS-PAGE.
REFERENCES
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