You treat a cell membrane preparation of the CHO cells provided with the enzyme

Question

You treat a cell membrane preparation of the CHO cells provided with the enzyme phospholipase C (PLC) at various concentrations of enzyme.

You collect supernatant and membrane samples after 30 minutes treatment and analyze them on an SDS-PAGEgel. Electrophoresed proteins are then transferred by immunoblotting to a membrane, and the membrane is probed with an antibody specific to p77 with an apparent molecular weight of 77.

You incubate membranes with a secondary antibody to visualize the specifically bound first antibody. You then analyze the immunoblot using densitometry and determine the relative levels of p77 in each sample (fold over basal lane 1 conditions), as shown below:

Amount of p77                                                                                 (Fold over lane 1)

Lane 1:
supernatant from cell membranes incubated
with saline buffer                                   1-fold

Lane 2:
supernatant from cell membranes incubated
with 1 micromolar PLC in saline buffer                            1-fold

Lane 3:
supernatant from cell membranes incubated
with 10 micromolar PLC in saline buffer                                         10-fold

Lane 4:
supernatant from cell membranes incubated
with 50 micromolar PLC in saline buffer                                         50-fold

Lane 5:
supernatant from cell membranes incubated
with 100 micromolar PLC in saline buffer                                        50-fold

Answer the following questions:

A) Why is it a benefit to use a primary AND a secondary antibody in the detection of the p77?

B) Why was it important to use varying concentrations of the enzyme to demonstrate specific action of the enzyme? What three observations are needed to determine whether a response is specific?

C) What type of protein might p77 be? Why?

Explanation / Answer

A) A primary and a secondary antibody would be specific for the protein of interest and hence would detect the protein in the Western blot.

B) Varying concentrations of enzyme will have different effect on the response. This can help us to ensure that the enzyme we are using is actually working and the result we are getting is not by chance and is a specific outcome of the enzyme used. The response would increase proportionately with each increase in the concentration and after a particular concentration their would be no change in the response which would suggest saturation.

C) p77 appears to be  inositol 1,4,5-trisphosphate (IP3) containing protein. Phosholipase C cleaves the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG remains bound to the membrane, and IP3 is released as a soluble structure into the cytosol. Since the protein is present in the supernatant fraction, it is most probably this protein.

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