Looking to characterize this novel protein. You are provided with: the human cDN
ID: 200393 • Letter: L
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: What type of protein might p18 be? Why?
Explanation / Answer
It is a membrane protein. As, phospholipase C breaks the protein lipid bond of cell membrane. The protein can be seen in the supernatant, this proves the fact. It is a translocator protein.
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