Experiment I – Co-precipitation of the EGFR and PLCg SH2 domain
GST-fusion proteins
Insert a fully labelled image of the Experiment 1 Coomassie-stained lower half of the protein gel, indicating the migration of the molecular weight markers and identifying any bands that you can.
Using the above gel image estimate the molecular weight, in kDa, of both the GST-SH2 fusion protein and GST by comparing to the molecular weight markers.
GST =
GST-SH2 =
From your above estimates what is the molecular weight of the SH2 domain?
SH2 =
What is the function and mode of action of IPTG and why did you add it to the bacterial culture?
Why does the pGEX vector have an ampicillin resistance gene and what protein does it code for?
EGF Receptor co-precipitation Experiment
Insert a fully labelled image of the Experiment 1 EGF receptor western blot, indicating the migration of the molecular weight markers and identifying any bands that you can.
What do these results indicate? (Interpret the combined results of the anti-EGFR western blot and Coomassie stained gel for each of the samples loaded on the gel)
In the protocol on Day 2 of the practical you are asked to make 35 mls of Triton Lysis Buffer using the recipe in the appendix. What volume of each of the stock solution components are you going to use to make this solution (show your calculations)?
2x TLB
TritonX-100
PAL
Benzamidine
Dithiothreitol
Sodium Orthovanidate
AEBSF
Additional volume of water
The Triton Lysis Buffer (TLB) you used to extract the cells in this experiment contains pepstatin, antipain and leupeptin (PAL). Why are these included in the lysis buffer and what do they do?
Sodium orthovanadate (Na3VO4) is also included in the TLB to inhibit a particular class of enzymes that might affect the experiment. What might sodium orthovanadate inhibit and why is it an important component of the buffer for these experiments?
What was the point of using GST as well as the GST-PLCg SH2 fusion protein in the precipitation (pull-down) experiment?
If you repeated the experiment with a mutant EGF receptor that could not bind ATP and was therefore inactive as a tyrosine kinase, what results would you expect to get (consider all lanes of the gel)?
Experiment II – Pathway analysis by western blotting with phospho-specific antibodies
EGF time course
Insert pictures of the four EGF time course blots below and fully label them, indicating the migration of the molecular weight markers. For each of the blots briefly comment on the following:
Can you identify any of the bands? If so, label them.
Are there changes in intensity with time?
What do you conclude from each blot?
Anti-phosphotyrosine (PY99)
Anti-phospho-ERK
Anti-phospho-PLCg
Anti-Tubulin
From the anti-phosphotyrosine blot, what do you think is the identity of the major band induced by EGF? How would you do to confirm this?
In the phospho-specific blots, the intensity of the bands decreases at later time points. Suggest a cellular mechanism for this and why is this important?
Why does the tubulin band not change in intensity in response to EGF? What is this type of blot used for?
