Quiz Topics for Protein Purification

Gel Filtration (how does it work)

Uses porous beads for the solid column matrix. The pores in the beads are large enough to allow for small proteins to pass through, but exclude large proteins, which flow around. Large proteins flow faster through the column compared to the small proteins, which take longer, slower paths through the numerous paths created by the pores.

Ion Exchange (How does it work)

Uses a charged column matrix to separate proteins based on their overall positive or negative charge [Anion (positively charged matrix)or Cation (negatively charged matrix) exchange chromatography can be used]. In a negatively charged matrix, a positively charged protein is attracted to negatively charged beads. Negatively charged proteins are repealed by the negatively charged beads and uncharged proteins flow through the column matrix unimpeded. In this situation, the negatively charged and uncharged proteins will move through the column quickly and the positively charged proteins will move through the column slowly. After the negatively charged and uncharged proteins flow through, an elution buffer (with sodium chloride containing positively charged sodium ions is added) which displaces the positively charged proteins from the beads and helps to elute them from the column.

Affinity (How does it work)

More specific. A ligand with a high affinity for the target protein is covalently bound to the gel matrix. The target protein will specifically bind to the matrix while other non-specific proteins flow through. As a result, the nonspecific proteins will move through the column much more quickly. To release the target protein an elution buffer containing an excess amount of a competing ligand is added. Target proteins can also be released by illusion buffers containing an altered pH or salt concentration. The competing ligand binds to the proteins, displacing them from the matrix.

Gel Filtration (How can the conditions be altered to change the rate of flow through the column or what binds to the column matrix)

Large proteins flow faster through the column compared to the small proteins, which take longer, slower paths through the numerous paths created by the pores. Change size of pores.

Ion-exchange (can the conditions be altered to change the rate of flow through the column or what binds to the column matrix)

In a negatively charged matrix, a positively charged protein is attracted to negatively charged beads. Negatively charged proteins are repealed by the negatively charged beads and uncharged proteins flow through the column matrix unimpeded. In this situation, the negatively charged and uncharged proteins will move through the column quickly and the the positively charged proteins will move through the column slowly. (Change matrix charge)

Affinity (can the conditions be altered to change the rate of flow through the column or what binds to the column matrix)

The target protein will specifically bind to the matrix while other non-specific proteins flow through. As a result, the nonspecific proteins will move through the column much more quickly. (change the affinity of bead to protein)

Bound Fraction

proteins that bound to the beads

Unbound fraction (flowthrough)

beads that flow through the chromatography

wash

binding something (ion exhange affintiy) assoication with matrix resign, whatever still in in bound fraction. High [] through random stuff stuck to beads. The wash removes the weakly bound things. (flow more buffer) (weak stuff removed) (rid of weakly associated things)

elution

buffer to get rid of bound fraction. Now you compete with something that binds to the matrix/change pH. (competitively binding to the beads/proteins.

When are the terms, bound fraction, unbound fraction (also called flowthrough), wash, and elution, as they apply to chromatography relevant?

Whether chromatography is done in a batch or column format, if proteins are binding to the matrix/resin, then these terms are relevant.

For gel filtration be familiar with considering whether proteins would come off the column in earlier or later fractions?

large proteins come out faster, smaller comes out in later fractions.

How does the SDS-PAGE work?

Proteins are treated with SDS. SDS denatures the folded protein and disrupts the secondary structure. Negatively charged SDS molecules coat the protein chain and give it a significant overall negative charge. SDS coats all proteins evenly so that the negative charge is proportional to the size of the protein and all proteins in solution will have the same charge-to-mass ratio. Moreover, because the proteins are denatured and form random coils, protein shape is not a factor in SDS-PAGE.

To ensure that the proteins in the SDS solution are fully denatured, the sample is heated in a boiling water bath. The sample is pipetted into a well in a polyacrylamide gel matrix.

An electric field is applied across the gel, which causes the negatively charged proteins to migrate across the matrix, attracted by the positively charged anode on the other side. While large proteins are restrained by the polyacrylamide gel, small proteins can move quickly. As a result, proteins become separated into bands within the gel according to size. The rate at which proteins move through the gel can be adjusted by changing the percentage of polyacrylamide in the gel. By measuring the observed migration distance of the unknown protein against that of known proteins, the molecular mass of the unknown protein can be determined.

Finally, the protein bands are not visible until the gel gets treated with a dye, such as Coomassie brilliant blue, which selectively stains proteins.

In an SDS-PAGE gel with staining, we can determine the molecular weight of the target protein by comparing it to a solution of marker proteins with known molecular masses.

Why is SDS used?

to denature the protein and give it a net negative charge

How does SDS affect what happens within the procedure?

SDS denatures the folded protein and disrupts the secondary structure. Negatively charged SDS molecules coat the protein chain and give it a significant overall negative charge. SDS coats all proteins evenly so that the negative charge is proportional to the size of the protein and all proteins in solution will have the same charge-to-mass ratio. Moreover, because the proteins are denatured and form random coils, protein shape is not a factor in SDS-PAGE.

Why would we want to use a high concentration of polyacrylamide?

A high concentration of polyacrylamide (increasing percentage of polyacrylamide) would decrease the rate of protein migration, and the separation between fast moving small proteins is improved at the expense of the large protein separation.

Why would we want to use a low concentration of polyacrylamide?

A low concentration of polyacrylamide (decreasing percentage of polyacrylamide) would increase the rate of protein migration, and the separation between fast moving large proteins is improved at the expense of the small protein separation.

2D gel electrophoresis

involves analyzing a protein sample by isoelectric focusing followed by SDS-PAGE to get one overall gel result.

Specific activity

…is a measure of the purity of a substance. It is defined as the activity per unit mass of the sample. as purity improves should have greater activity.

How would Specific activity be relevant for tracking whether a purification procedure worked well?

….is a powerful tool for monitoring the effectiveness of a purification procedure because it directly reflects the enrichment of the desired molecule relative to other contaminants. In summary, a successful purification procedure will show a progressive increase in the specific activity of the target molecule as contaminants are removed.

Why does adding a salt elute samples off of a cation or anion exchange column? Would adding salt also be relevant for the other two methods

Salt interferes with the electrostatic interactions. Competitive interaction with the matrix proteins. Salt does not affect Gel but may affect affinity due to the noncovalent interactions with a ligand.

Why do all proteins migrate in the same direction? (SDS-PAGE)

All same charge (SDS makes them have the same negative charge)

How does changing the polyacrylamide concentration influence migration? iffy

Makes the smaller proteins have better separation, make the bigger proteins have less separation (slowly finding way down)

Isoelectric focusing can determine….

….Isoelectric point, or pI, of proteins. The same protein at different pH values will have a different net charge value. If at pH < pI, migrates to cathode. If at pH > pI, migrates to anode.