Question

when perfoming the Isolation of the GFP protine lab, what should be present in the collection tubes after each step in the column chromatography? explain in detail please.

Answer 1

The green fluorescent protein (GFP) is a protein that exhibit bright green fluorescence when exposed to blue light. GFPSparkTM is an improved variant of the green fluorescent protein GFP. It possesses bright green fluorescence (excitation/ emission max = 487 / 508 nm) that is visible earlier than fluorescence of other green fluorescent proteins. GFPSparkTM is mainly intended for applications where fast appearance of bright fluorescence is crucial. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. It is specially recommended for cell and organelle labeling and tracking the promoter activity.

Chromatography is a term that describes a huge and diverse set of methods for

separating molecules based on their different properties. In it’s simplest form, it consists of

three steps:

1) Binding. A mixture of molecules is exposed to a solid material. The molecule of

interest binds to the solid via some non-covalent interaction; the others do not.

2) Wash. The solid is rinsed with a solution (wash buffer) which allows the molecule of

interest to remain bound to the solid while washing off the unwanted molecules.

3) Elution. The solid is rinsed with a different solution (elution buffer) which releases the

molecules of interest from the solid. The resulting solution contains (ideally) only the

molecule of interest.

In this lab, we will use the fact that GFP has significantly more hydrophobic amino

acids on its surface than most proteins. Therefore, it will bind - via the hydrophobic effect - to

a solid that also has a hydrophobic surface more tightly than most other proteins.

We will also use another important property of the hydrophobic effect to selectively

bind and elute GFP. In solutions with a high salt concentration, the hydrophobic effect is

strengthened; in solutions with low salt concentrations, the hydrophobic effect is weakened. It

turns out that it doesn’t matter what the salt is; we will use ammonium sulfate ((NH4)2SO4)

because it is very soluble in water and does not harm most proteins.

To make the process easier to carry out, the solid material called the “resin” will be

packed into a tube called a chromatography column. Here’s how it will work (the hydrophobic

solid resin is indicated by circles; GFP is indicated by black triangles; other molecules are

indicated by the other shapes):

0) Equilibration – a solution containing the same buffer as Step (1) is run through the

column to prepare it for chromatography (not shown above).

1) Binding - a solution containing the cell contents, including GFP, is run through the

column in high salt. GFP binds via the hydrophobic effect; most other molecules don’t.

Those that don’t stick flow through the column and are discarded.

2) Wash - a solution of medium salt is run through the column. GFP remains bound

because it has more hydrophobic amino acids on its surface while any remaining other

molecules wash out. The molecules in the wash are discarded.

3) Elution - a solution of low salt is run through the column. This disrupts the

hydrophobic interaction between the column and GFP so GFP comes off the column in

relatively pure form for study