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18 Dec 2019

How does HIC column function to purify GFP frm most proteins inthe cell?

How is GFP used in molecular biology?

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31 Dec 2019
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Related questions

You have purified protein you call MAP15. MAP 15 binds totubulin that is GTP-bound tubulin and does not bind to tubulin thatis GDP-bound. You genetically engineered cells to express a greenfluorescent protein (GFP) –tagged version of MAP15. This GRP-MAP15fusion protein does not appear to disrupt the function of MAP15.You use a red fluorescent dye to label the microtubules so that youcan visualize GFP-MAP15 and the microtubules at the same time usingfluorescence microscopy (Note that the red dye does not appear toaffect microtubule structure and function).

A. If you were to polymerize some red-labeled microtubules in adish, would you expect GFP-MAP15 to be associated with one or bothends of a microtubule, with the middle of the microtubule, or beevenly distributed throughout the microtubule? Explain.

B. You examine the red microtubules and GFP-MAP15 in a cell ininterphase. Do you think that you will see GFP-MAP15 associatedwith all microtubules? Explain.

C. If you were to take time-lapse images in which you visualizeonly GFP-MAP15 (and not microtubules), what would you expect tosee? Describe what you think would happen to GFP-MAP over time withrespect to its localization inside of this cell.

Please answer all of the following questions with the best answer.

1.) In Hydrophobic interaction Chromatography, why would we use TE buffer to push out the GFP (green fluorescent protein)?
a.)TE has a low salt concentration, allowing the GFP to refold
b.) the EDTA component of TE inhibits many proteases
c.) the Tris component of TE inactivates the HIC beads

2.) In column chromatography the bed volume is influenced by:
a.) the eluent being applied to the column
b.) the stationary phase
c.) the temperature
d.) the radius of the column

3.) What is the predicted result of increasing amounts of lignin is added to a cellulobiase reaction?
a.) cellulobiase becomes denatured
b.) more product is made
c.) less product is made
d.) Vmax of cellulobiase is decreased

4.) Polypeptides with a hydrophobic core can best be denatured by which of the following treatments?
a.) adding 2M of NaCl to the solution
b.) addition of distilled water
c.) resuspension in TE buffer
d.) multiple freeze-thaw cycles

5.) Intense sound waves can be used to break down bacterial cell walls without damaging he protein of interest.
a.) true
b.) false

6.) Bacteria can be used to make eukaryotic protein.
a.) true
b.) false

7.) pH changes will affect the vicibility of p-nitrophenol.
a.) true
b.) false

8.) A lysozyme pokes holes in a cell wall making it easier for protein to be released from bacteria.
a.) true
b.) false

9.) Bacteria will store excess protein in vesicles.
a.) true
b.) false
Please answer all of the following questions with the best answer.

1.) In Hydrophobic interaction Chromatography, why would we use TE buffer to push out the GFP (green fluorescent protein)?
a.)TE has a low salt concentration, allowing the GFP to refold
b.) the EDTA component of TE inhibits many proteases
c.) the Tris component of TE inactivates the HIC beads

2.) In column chromatography the bed volume is influenced by:
a.) the eluent being applied to the column
b.) the stationary phase
c.) the temperature
d.) the radius of the column

3.) What is the predicted result of increasing amounts of lignin is added to a cellulobiase reaction?
a.) cellulobiase becomes denatured
b.) more product is made
c.) less product is made
d.) Vmax of cellulobiase is decreased

4.) Polypeptides with a hydrophobic core can best be denatured by which of the following treatments?
a.) adding 2M of NaCl to the solution
b.) addition of distilled water
c.) resuspension in TE buffer
d.) multiple freeze-thaw cycles

5.) Intense sound waves can be used to break down bacterial cell walls without damaging he protein of interest.
a.) true
b.) false

6.) Bacteria can be used to make eukaryotic protein.
a.) true
b.) false

7.) pH changes will affect the vicibility of p-nitrophenol.
a.) true
b.) false

8.) A lysozyme pokes holes in a cell wall making it easier for protein to be released from bacteria.
a.) true
b.) false

9.) Bacteria will store excess protein in vesicles.
a.) true
b.) false

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