Experiment 7: Introduction to Protein Column Chromatography Flashcards
What is chromatography?
Chromatography is the name given to a broad selection of processes that are designed to separate different molecules from each other
What is the role of the stationary phase?
The role for the stationary phase is that it will interact to varying degrees with different molecules in the mobile phase that are moving past it.
What happens if a molecule interacts strong with the stationary phase? what if it interacts weakly?
If a molecule in the mobile phase interacts strongly with the stationary phase, it will spend a certain portion of its time immobilized on the stationary phase material.
Conversely different molecules which interact weakly or not at all with the stationary phase will spend more time moving.
What is ion exchange chromatography (IEX)? Why is it useful?
IEX is based upon reversible electrostatic interactions between charged molecules and charged functional groups bound to inert resin beads packed in a column.
IEX is a useful protein purification method bc different proteins have different amino acid compositions, and therefore different proteins will have different numbers of positively or negatively charged amino acid side chains at any given pH.
What is affinity chromatography? what is a challenge with AC?
AC is based upon the specific and reversible high affinity interaction between a protein and a ligand molecule that is covalently linked to an insoluble solid support packed into a column.
The biggest challenge to using affinity chromatography techniques is finding a ligand that will bind to your protein but not other proteins as this is the key for the specificity of the technique.
What are tags?
Proteins can be synthesized to include sequences of amino acids which are known to bind to ligands that can be easily attached to a column. These additional sequences added to proteins are called tags.
What is the most common tag?
His-Tag. A His-Tag sequence is typically a sequence of six consecutive histidine residues that is attached to the beginning or the end of a protein sequence. Proteins with His-Tags will bind to columns that have nickel ion immobilized on the column.
How do you get the bound protein out of the column (might be specific to AC)?
The most common way is to add a high [ ] of another molecule that also has the ability to bind to the protein of interest.
Since the protein bound to the column also has the ability to bind to the ligand that has been added to the column, the high [ ] of the added free ligand out-competes the column for binding to the protein with the result that the protein elutes from the column and it can be collected in a fraction relatively pure.
What is size exclusion chromatography (SEC)?
size exclusion chromatography is a method that separates molecules on the basis of their size. Unlike the previous two methods, the beads for this form of purification do not have function groups attached to them. Instead the beads are porous and filled with channels of varying sizes.
How does SEC work?
When a mixture is applied to a column filled with size exclusion beads and allowed to sink into the column, molecules may or may not be able to enter the pores and channels w/n the beads depending on how but the molecules are. small molecules can enter the bead, move around in the bead before exiting the bead and returning to the general flow of the mobile phase.
any molecules that cannot enter into any beads will elute first, and smaller molecules will elute later according to their size with he smaller molecules eluting last.
What are the two reasons why SDS-PAGE can separate proteins on the basis of size?
1- during the preparation of the proteins for loading on the gel they all are structurally altered to give them the same q/f ratio
2- they are electrophorized through a sieving porous structural support which we are calling gels.
Why do we heat a protein sample before loading it on to the SDS-PAGE?
the three dimensional structure of the protein is destroyed by the high temperature. This allows the SDS to associate with the peptide bonds of the protein at a constant ratio. Since the SDS carries a -ve charge, the denatured protein is now covered with -ve charges that greatly exceed the original charge on the protein.
