Myers:EGFR IP

Overview

Purification of EGFR for Proteomics Analysis using Cetuximab

Materials

Human cells expressing EGFR

NETN (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Igepal, and 5%glycerol)

Cetuximab (monoclonal antibody to EGFR also used as an EGFR antagonist)

Protein A/G suspension (Pierce)

Cell Culture: A431 cells (~70% confluent) were serum-starved (24hrs) and either treated only with 40nmol EGF for 20 min or pre-incubated with 10ug/ml cetuximab inhibitor and stimulated with EGF. Cells were harvested on ice with Mg, Cl-free PBS with added phosphatase inhibitor cocktail. Cells were spun @ 1000rpm and flash frozen in liquid nitrogen.

Procedure

EGFR IP:

1. Cell pellets lysed on ice in 5mL NETN buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Igepal, and 5%glycerol) containing protease inhibitors (1mM phenylmethyl-sulfonyl fluoride, 1-ug/ml leupeptin, 1-ug/ml pepstatin, and 1-ug/ml aprotinin) and 1x MSRC phosphatase inhibitor cocktail for 30 min. (2 plates = 381-385 ug/ul)
2. Cell suspensions were sonicated for 30 sec pulses 3x and lysates cleared by centrifugation at 8000 rpm for 10 minutes.
3. ~190ug of lysate (-500ul) was aliquoted and combined with LDS loading buffer and DTT (@ final concentration of 50 mM)  Input.
4. ~1.7mg of lysate (in 4.5mL) was transferred to clean tubes (1.5 mL/tube) and cetuximab antibody was added at a ratio of 5ug Ab to 1mg of lysate.
5. Antibody and lysate were incubated 4C for 1.25hrs.
6. 30ul of (pre-equilibrated) Protein A/G resin suspension and incubated with the lysate for 45min @4C.
7. The Suspension was centrifuged @2000rpm (~0.5xg) for 2 min @4C (500uL was removed and prepared similar to input from step 3).
8. Resin washed 3x with NETN buffer by inverting tube 15 times. Buffer was washed with 500x bead volume.
9. Eluted in 2XLDS and DTT( @ final concentration of 50mM) by heating for 5min at 80C.
   • always keep lysates on ice and do not introduce bubbles during sample preparation as the surface tension can denature proteins.

Notes

1. Follow standards for protein handling and immunoprecipitation.

References

Relevant papers and books

1. ImClone Systems Incorporated, New York, NY 10014 USA and Bristol-Myers Squibb Company, Princeton, NJ 08543 USA; All rights reserved. ERBITUX is a registered trademark of ImClone Systems Incorporated.

Contact

• Jeremy Myers

--Jeremy S. Myers 11:33, 10 April 2008 (EDT) or instead, discuss this protocol.

Post questions or comments on my talk

http://openwetware.org/wiki/User_talk:Jeremy_S._Myers