The green fluorescent protein GFP is normally a cytosolic protein. You have gene
ID: 97992 • Letter: T
Question
The green fluorescent protein GFP is normally a cytosolic protein. You have genetically engineered a form of GFP that contains a KDEL sequence. When the construct containing this new protein (GFP with KDEL) is transfected into normal human fibroblasts and examined using fluorescence microscopy, the fluorescence appears throughout the cytoplasm, as drawn on the diagram on the right.
A. How would you explain this pattern given that KDEL is supposed to be an ERspecific sorting sequence?
To analyze the results further, fractions of different organelles and the cytoplasm were collected from cells expressing this KDEL-containing GFP construct and then examined on Western blots using antibodies against GFP (27 kDa) and protein disulfide isomerase (PDI), a protein that is normally found in the rough ER (RER) and that is approximately 55 kDa. The blot confirms the presence of GFP exclusively in the cytoplasm, and as expected a PDI signal in the RER fraction.
B. How would you explain the PDI band, albeit weak, in the Golgi fraction?
C. Given the function of PDI proteins, what would you expect if both alleles of a PDI gene were knocked out in mice? [Think about what they do and how essential they are, if at all] The antibodies used above do not detect any signals in the nuclear fraction, which indicates their specificity or the fact that no proteins were isolated and loaded in the nuclear fraction lane.
D. What antibody could be used to show there were nuclear proteins present in this sample? [Just think of an appropriate protein that you could make an antibody against. There are many correct answers]. Justify your choice.
JUST QUESTION D. thank you
Explanation / Answer
D) Antibodies that can be used to show the presence of nuclear proteins in a sample are,
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.