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Revision as of 11:33, 10 April 2008
Purification of EGFR for Proteomics Analysis using Cetuximab
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.
- 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)
- Cell suspensions were sonicated for 30 sec pulses 3x and lysates cleared by centrifugation at 8000 rpm for 10 minutes.
- ~190ug of lysate (-500ul) was aliquoted and combined with LDS loading buffer and DTT (@ final concentration of 50 mM) Input.
- ~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.
- Antibody and lysate were incubated 4C for 1.25hrs.
- 30ul of (pre-equilibrated) Protein A/G resin suspension and incubated with the lysate for 45min @4C.
- The Suspension was centrifuged @2000rpm (~0.5xg) for 2 min @4C (500uL was removed and prepared similar to input from step 3).
- Resin washed 3x with NETN buffer by inverting tube 15 times. Buffer was washed with 500x bead volume.
- 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.
- Follow standards for protein handling and immunoprecipitation.
Relevant papers and books
- 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.
- Jeremy Myers