Anti-Myc tag antibody (Agarose) (ab1253)

Is there an IP protocol that should be used with this antibody? Specifically, customer would like to know if the beads should be washed prior to use and if so, with what buffer?

ANSWER:

Thank you for your enquiry.

You don't need to wash the beads prior to using, and perform the IP at 4C. Also, use 15-25 ul of gel slurry per 0.1 to 1 mg of protein lysate or extract.

Below is a general IP protocol that the originator of this antibody sent that you can use as a guideline. If you have any additional questions, please contact us again.

Immunoprecipitation Protocol For use with Antibodies Immobilized on Agarose:

A. To microcentrifuge tube add: 1) 15 to 25 ul of slurry of agarose immobilized Ab (3.75 to 6.125 ug Ab)

2)500 ul 2x Stringent IP buffer*:

100 mM Tris-HCl (pH 8.0) 1000 mM Nacl

Chelating agents: 2 mM EDTA and/or 2 mM EDTA

Detergents: 2 % IGEPAL CA-630 (replacement for NP40) 0.2 % SDS 2 % Deoxycholate

Protease Inhibitors: 20 ug/ml Aprotinin 20 ug/ml Leupeptin 2 mM PMSF or 10 ug/ml Pepstatin A or 8 mM AEBSF

Phosphatase Inhibitors – If Necessary: 200 mM NaF 2 mM Na3VO4 50 mM ?-Glycerophosphate

3)Cell lysate (0.1 to 1.0 mg total protein)

4)diH2O to a total volume of 1 ml

B. Vortex and incubate for between 1 and 24 h at 4 deg. Turning tubes on a rotating platform wheel allows continuous mixing.

C. Centrifuge (16,000 x g, 3 to 5 min), aspirate and discard supernatant

D. Wash pellet in 1x IP buffer 3 to 6 times. Washing consists of resuspending pellet, centrifuging (16,000 x g, 3 to 5 min), aspirating and discarding supernatant.

E. After last wash, resuspend pellet in 30 ul 2x Electrophoresis Sample Buffer, boil 5 min.

F. Centrifuge, load supernatant onto SDS-PAGE gel, electroblot onto PVDF membrane and perform Western Blot Analysis.

*Buffers for Immunoprecipitation Assays

For analysis of a single protein without associated proteins, the use of high ionic strength, strong detergents, and rigorous washing of the immunoprecipitate are recommended. One suitable buffer is the Strigent IP buffer outlined above.

For co-precipitation of associated proteins, the use of near physiological ionic strength and gentle detergents is recommended. One suitable buffer is the Lenient IP buffer outlined below.

The buffers outlined represent two extremes. The optimal buffer for ones given application must be empirically defined. This buffer may be of intermediate ionic strength and/or detergent composition.

1x Lenient IP buffer:

50 mM Tris-HCl (pH 8.0) 100 mM Nacl

Chelating agents: 1 mM EDTA and/or 1 mM EGTA

Detergents: 0.5 % IGEPAL CA-630 (replacement for Nonidet P40) Protease Inhibitors: 10 ug/ml Aprotinin 10 ug/ml Leupeptin 1 mM PMSF or 5 ug/ml Pepstatin A or 4 mM AEBSF

Phosphatase Inhibitors – If Necessary: 100 mM NaF 1 mM Na3VO4 50 mM ?-Glycerophosphate