Lysates of cell lines and xenograft tissues were generated using lysis buffer as previously reported (25 (link)). The lysate was centrifuged at 16,000 g at 4°C for 15 min. 40 micrograms of total protein for each sample were separated by 8–10% SDS-PAGE and transferred onto a Westran S membrane (Whatman Inc. Floham Park, NJ). Desired proteins were probed with corresponding antibodies. Rabbit anti-human AKT, ERK, pAKT, pERK, EGFR, pEGFR, HER2 pHER2, HER3 and pHER3 antibodies (1:1000 dilutions) were purchased from Cell Signaling (Danvers, MA), mouse anti-human β-actin (1:10000 dilution) from Sigma (St. Louis, MO), anti-human EGFR and HER3 antibodies from Santa Cruz (Santa Cruz, CA), and anti-human pEGFR antibody from Millipore (Temcula CA). HRP-conjugated secondary anti-mouse and anti-rabbit IgG (H+L) was obtained from Promega (Madison, WI). Bound antibody was detected using the SuperSignal West Pico Chemoluminescence system (Pierce, Inc., Rockford, IL). Image J software was used for blot quantification. Protein densitometry was determined by Image J software.
Protocol detail
Protein Expression Analysis in Cell Lines
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mouse anti-human β-actin
anti-human EGFR and HER3 antibodies
anti-human pEGFR antibody
HRP-conjugated secondary anti-mouse and anti-rabbit IgG (H+L)
Actin
Anti antibody
Anti igg
Antibodies
Antibody
Buffer
Cell lines
Densitometry
Dilution
Egfr
Her2
Her3
Human
Membrane
Mouse
Promega
Protein
Rabbit
Sds page
Tissues
Xenograft
Corresponding Organization : Emory University
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Variable analysis
independent variables
- Lysates of cell lines and xenograft tissues were generated using lysis buffer
- Levels of AKT, ERK, pAKT, pERK, EGFR, pEGFR, HER2, pHER2, HER3 and pHER3 proteins
- Centrifugation of the lysate at 16,000 g at 4°C for 15 min
- Loading of 40 micrograms of total protein for each sample
- Separation of proteins by 8–10% SDS-PAGE
- Transfer of proteins onto a Westran S membrane
- Use of specific antibodies at 1:1000 (for AKT, ERK, pAKT, pERK, EGFR, pEGFR, HER2, pHER2, HER3, pHER3) and 1:10000 (for β-actin) dilutions
- Use of HRP-conjugated secondary anti-mouse and anti-rabbit IgG (H+L) antibodies
- Detection of bound antibody using the SuperSignal West Pico Chemoluminescence system
- Use of Image J software for blot quantification and protein densitometry
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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