Question

For the experimental background, we treated 3T3 mouse fibroblasts with EGF and are now trying to detect the phosphorylated tyrosine proteins using primary and secondary antibodies as stains. We also made 4 different samples (a control with no EGF, a sample exposed for 1 minute to EGF, a 5 minute, and a 10 minute).

1) What does the appearance of a new band in a western blot mean?

2) When a band on a western blot increases in density over time, what does it mean?

3) When a band on a western blot decreases in density over time, what does it mean?

4) As it relates to EGF signaling, what process in the cell would possibly lead a band to increase in density over time?

5) As it relates to EGF signaling, what process in the cell would possibly lead to a band to decrease in density over time?

6) Why would a control (or 0 time) sample show bands for tyrosine phosphorylated proteins?

Thank you!

Answer 1

1) As the antibodies can only bind to the protein of interest, only one band should be visible. This band indicates the expreesion of protein of interest.

2) The thickness of the band corresponds to the amount of protein present. More dense band indicates the expression level of particular protein is high. The density increase over time means the expression of particular protein increased over time.

3) When a band on a western blot decreases in density over time, that means the expression of particular protein decreased over time. Exposure with EGF over time can inhibit expression of particular protein.

4) EGF is one of the high affinity ligands of EGFR. EGF siganling control various biologic responses such as proliferation, differentiation, cell motility, and survival. Increased band density over time indicates that the trans-membrane proteins are activated following binding with ligands such as epidermal growth factor (EGF).