Protein purification Flashcards
What are the overall steps of protein purification?
Determine the SOURCE of protein for purification, cell LYSIS, protein PURIFICATION, and protein ASSAY to check purity levels and concentration.
What are the possible sources of protein for purification?
Protein can be sourced from tissue as endogenous protein or be cloned as a recombinant protein in bacteria/insect cells/mammalian cells. Common recombinant protein is from E.coli.
Why is E.coli used as a source of recombinant protein?
E.coli is EASY to grow and GROWS FAST with high cell count, TRANSFORMATION with the protein of interest is fast and easy, RICH MEDIA to culture E.coli is readily available and cheap.
How is cell lysis performed in protein purification?
Cells can be lysed by FREEZE THAWING (liquid nitrogen) at -20C to burst open the cell membranes, use NON-IONIC DETERGENT (Triton-X) to solubilize the membrane, SONICATION to disrupt the cell membrane, or ULTRACENTRIFUGE to collect the protein in the supernatant and cell membrane in the pellet.
What are examples of protein purification steps? To separate the POI from other cytosolic proteins.
Affinity chromatography, Gel filtration (size exclusion) chromatography, ion exchange chromatography, differential solubility with ammonium sulphate, isoelectric focusing, SDS-PAGE with western blotting.
How does ammonium sulphate work as a protein purification step?
Ammonium sulphate is used as an initial purification step based on differential solubility. It displaces water molecules to precipitate proteins with its high salt concentration, causing proteins to bind to each other and precipitate. But the salt needs to be removed before the next purification step.
What purification step can be conducted in high salt concentrations after using ammonium sulphate to precipitate proteins?
Gel filtration chromatography (size exclusion) and hydrophobic interaction chromatography.
What methods can be used to remove salt during protein purification?
Dialysis, diafiltration, gel filtration chromatography.
How does dialysis remove salt from the sample?
In dialysis, the sample is placed in a bag with pores too small to allow the POI to pass but big enough for salt ions. Several BUFFER EXCHANGES will remove the salt ions from the sample.
How does DIAFILTRATION remove salt from the sample?
Diafiltration will permeate the salt through a PRESSURE-DRIVEN FILTRATION MEMBRANE and retain the POI. This also concentrates the protein.
How does gel filtration remove salt from the sample?
Gel filtration chromatography uses RESIN with pores that salt ions can enter while proteins which are larger cannot and are carried with the buffer to retain the proteins.
How does affinity chromatography work to precipitate the POI?
Affinity chromatography uses an AFFINITY MOLECULE (Nickel) that is bound to a SOLID support (beads/column). The sample flows into the column and the POI strongly binds with the affinity molecule on the beads, then the beads are CENTRIFUGED & WASHED or washed several times with INCREASING NaCl CONCENTRATION to remove unbound proteins. The POI can be eluted with a molecule (imidazole) that COMPETES FOR BINDING WITH THE AFFINITY MOLECULE to displace the POI.
How does gel filtration chromatography work to purify proteins?
Gel filtration chromatography separates proteins by SIZE with a column filled with POROUS RESIN. Larger proteins will not enter the pores and flow through quickly and elute first while smaller proteins are eluted later. Multiple fractions are collected into tubes until all proteins are eluted out with INCREASING VOLUMES OF BUFFER. SDS-PAGE with western blotting can be performed after to detect POI.
What factors affect gel filtration chromatography?
SHAPE & AMOUNT of protein, LENGTH of the column (longer separates better), and the PORE SIZE of resin.
How does isoelectric focusing work to purify proteins?
isoelectric focusing separates proteins based on their charge and isoelectric point (pI) which is the pH where they have a neutral charge. Proteins are loaded onto a gel with a STABLE PH GRADIENT and an ELECTRIC FIELD to allow proteins to MIGRATE to their isoelectric point and stay there. The GEL STRIP CAN BE LOADED onto an SDS-PAGE and stained with COOMASSIE BLUE to detect the POI (2D electrophoresis to see molecular weight and pI of the proteins).
