Question

Part B. Working as a Biology 499 student, your supervisor would like to examine the protein- protein interaction in the tyrosine kinase complex as shown in the following figure. Specifically, you are asked to show the direct interaction between Sos, Ras and GRB2, in the presence or absence of EGF. Sinding of CAB2 and Sou couples recrgeo to inacie Two separate dishes of cells were each transfected with a different type of Vector DNA: HA GST, HA-Sos. After growing for 7 days, each dish was divided into two; and EGF was added to one of them. Cells from each sample were harvested and whole cell lysates were subjected to immunoprecipitations (IP) by the use of anti-HA antibodies and Protein A agarose beads. Predict the results in the following immunoblots when anti-HA (upper panel), anti-GRB2 (middle panel) and anti-Ras antibodies (lower panel) were used. (Note: the size of GST is 26kDa, Sos is 70 kDa, GRB2 is 25kDa and Ras is 21kDa) (6 marks) P: HA EGF+ B: HA IB: GRB2 IB: Ras

Answer 1

The blots with the following bands will be obtained as given below in the diagram--

IP: HA EGF: B: HA IB: Ras

In the first lane EGF is not added and HA-GST construct is transfected, therefore will be light HA band  obtained after blotting with the anti-HA antibodies after performing the immunoprecipitation in the cell lysate.

In the second lane EGF was added to the cell lysate along with the transfection of the cell line with HA-GST construct leading to expression of HA protein inside the cell line. After immunoprecipitation with the anti HA antibody, the band of HA will be obtained in the cell lysate.

In the third lane, EGF was not added and the HA-Sos protein construct was transfected in the cell line. Therefore after performing the process of immunoprecipitation with the anti-Sos antibody, there will be a light band of sos protein in the blot obtained from the cell lysate.

In the fourth lane, EGF was added to the cell line and HA-sos protein was transfected in the cell line. Therefore, after performing the process of immunoprecipitation with anti-sos protein, a dark band of sos protein will be observed in the western blot analysis.

In the first lane, EGF was not added and HA-GST construct was transfected in the cell line. Therefore after immunoprecipitation with anti-Grb 2 protein, there will be a light faint band of Grb2 protein observed on the western blot.

In the second lane, EGF was added to the cell line and HA-GST construct was transfected in the cell line. Therefore after immunoprecipitation with anti-Grb2 protein, there will be a strong band of Grb2 protein which will be observed on the western blot.

In the third lane, EGF was not added and HA-Sos construct was transfected in the cell line. Therefore after precipitation with anti Grb2 protein , light faint band of protein will be observed in the western blot.

In the fourth lane, EGF was not added and HA-Sos construct was transfected in the cell line. Therefore after immunoprecipitation with the anti-Grb2 protein, there will be formation of strong protein band in the western blot.

In the first land EGF was not added and HA-GST construct was transfected. After immunoprecipitation with the anti-HA antibody, there will be observation of a light protein band in the blot.

In the second lane, EGF was added and HA-GST construct was transfected. After immunoprecipitation with the anti-HA antibody, there will be observation of a strong protein band in the blot.

In the third lane, EGF was not added and HA-Sos construct was transfected. After immunoprecipitation with the anti-Grb2 antibody, there will be observation of a light faint band in the blot.

In the fourth lane, EGF was added and HA-Sos construct was transfected. After immunoprecipitation with anti-Grb2 antibody, there will be observation strong band of antibody in the blot.

In the first lane, EGF was not added and HA-GST was transfected in the cell line. After immunoprecipitation with anti-ras antibody a light band of protein was observed in the blot.

In the second lane, EGF was added and HA-GST was transfected in the cell line.After immunoprecipitation with anti-ras antibody a strong band of protein will be observed.

In the third lane, EGF was not added and HA-Sos protein construct was transfected in the cell line. After immunoprecipitation with anti-ras antibody, a light faint band of Ras protein will be obtained in the blot as protein activated is Sos protein due to formation of EGF-Grb2-Sos protein with Ras-GDP activation to Ras-GTP in the cell membrane.

In the fourth lane, EGF was added and HA-Sos construct was transfected in the cell line. After immunoprecipitation with anti-ras antibody, a strong band of protein will be obtained in the blot due the activation of EGF-GRb2-Sos protein complex in the membrane of the cell line leading to the process of phosphorylation of EGF receptor at the tyrosine motif.

HA-GST is the preclearing complex in the cell lysate which can be immunoprecipitated with the protein A beads to remove the non specific proteins which are pulled down in the cell lysate as HA has immunoaffinity for some proteins present inside the cell.