Looking to characterize this novel protein. You are provided with: the human cDN

Question

Looking to characterize this novel protein. You are provided with: the human cDNA corresponding to the protein, a Chinese Hamster Ovary (CHO) cell line expressing the cDNA for the p18, and the amino acid sequence (using the one-letter code) . Shown following is the first 30 amino acid region of this putative amino acid sequence for the target: 1YFNPS 6TSDWPT 11LAPAN 16YTFLF 21FLARY 26WYINL30

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-PAGE gel. Electrophoresed proteins are then transferred by immunoblotting to a membrane, and the membrane is probed with an antibody specific to the protein with an apparent molecular weight of 18. 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 p18 in each sample (fold over basal lane 1 conditions), as shown below:

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: 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?

Explanation / Answer

Increasing Enzyme Concentration will increase the rate of reaction, as more enzymes will be colliding with substrate molecules. So it is important to use varying concentration of enzyme to demonstrate the specific action of the enzyme.

Observations needed:

Detecting antigen and antibody (first antibody) interaction, for detecting and quantitating p18 protein.

Detecting antibody-antibody interaction (first antibody and second antibody) for removing the unbinded first antibody.

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