Chromatography Flashcards
What are the different chromatographic separation techniques?
-Salting in or out:
*Salting in: lower the concentration of your buffer, add
salt and you can separate the sample.
*Salting out: Increase concentration of your buffer and
the protein will be precipitated
-Column chromatography
*Ion exchange
*Gel filtration or size exclusion
*Affinity
*Reverse-phase HPLC (high performance liquid
chromatography)
-Electrophoresis
-Isoelectric focusing
What happens to the sample when it is being dissolve?
The sample is dissolved in mobile phase (gas,liquid) and then forced through the stationary phase.
What does the solubility of the protein depend on?
Protein contains multiple charged groups; its solubility depends on the concentrations of dissolved salts, the polarity of the solvent, the ph, and the temperature. Some or all of these variables can be manipulated to selectively precipitate certain proteins while others remain soluble.
What is the difference between salting in and salting out?
- The solubility of a protein at low ion concentrations increases as salt is added, a phenomenon called “salting in”.
- In contrast to salting in, salting out occurs in aqueous solutions of high ionic strength that reduce the molecule’s solubility causing certain proteins to precipitate
- Ideally, the type of salt being used and the concentration of the salt can be varied to selectively precipitate the molecule
What is chromatography?
- Chromatography is a separation method in which components partition between a moving phase and a stationary phase.
- All techniques of chromatography utilise different properties of the proteins;
- Charge distribution
- Molecular size
- Solubility
- Binding properties
- Combining two properties means it is possible to derive a purification protocol for any protein.
- Chromatography involves a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical fluid).
- The mobile phase is then forced through an immobile, immiscible stationary phase.
- The phases are chosen such that components of the sample have differing solubilities in each phase.
What is column chromatography?
Where you have the protein placed in a column that contains special substances to allow separations- called cation exchangers.
How does cation exchange chromatography work?
-Column with the negatively charged matrix – usually carboxymethyl cellulose
-The positive charge will bind to negative column – the rest will flow through in the following order.
-Proteins bind to an ion exchanger with different affinities.
-Column is washed with buffer, to elute proteins based on relatively low affinities for the ion exchange resin.
*The greater the binding affinity of a protein for the ion
exchange column, the slower it will be eluted off the
column i.e. the more negative net charge the faster it
will move down the column
*The four elutions will have large net negative charge,
net negative charge, net positive charge and large
net positive charge in that order
*Elution can also be determined by salt concentration
of buffer and/or pH of the buffer (stepwise elution or
gradient elution)
*And so elution can determined by salt concentration
of buffer, ionic strength of buffer or the pH of the
buffer.
What is ion exchange chromatography (IEX)?
-The property used is charge distribution of the protein
-Often first method to be done
-Separates larger amounts of materials than gel filtration
-The stationary phase is polymer (matrix/resin) attached with charged groups (negative or positive depending on what protein is being separated)
*Acidic gps of resin interact with positively charged
proteins and are called cation exchangers.
*If the protein is negatively charged then it is anion
exchange
How does ion exchange chromatography work using stepwise elution?
- The sample is inserted. The column is washed with a salt with low salt content. The weaker proteins that do not bind to the column will elute first.
- Then a salt with medium concentration is used and then with high concentration. The proteins are eluted based on their affinity to the column.
What is gel filtration chromatography (GFC)?
- Also called size exclusion chromatography or molecular sieve chromatography
- This chromatography separates proteins based on their size and shape- large proteins elute first. This is widely used to purify biological molecules in complex samples such as blood.
- The column consists of porous matrix by using porous beads of dextran or agarose - Sephadex and Sepharose are commonly used commercial preps.
How does gel filtration chromatography work?
- If we assume proteins are spherical in shape with different sizes.
- The smaller proteins will go into the pores of the matrix. The big ones cannot go through the pores.
- Whilst you are doing this the column is washed with buffer ensuring the proteins are eluted.
- Elution volume (Ve) is the volume of a solvent required to elute a given solute from the column.
- If a protein has a small molecular size more washing with the buffer is required as so small proteins have a higher elution volume.
- GFC is generally used near the end of the purification process e.g. to separate the correctly folded native protein from the denatured protein.
- Sample put through the column
- Washed with buffer
- Bigger molecules will come out first
- Smaller ones come out last.
What is affinity chromatography (AC)?
- Many proteins can bind specific molecules very tightly but non-covalently to the column. This property can be used to our advantage as AC is used to isolate antibodies, antigens, hormones and other proteins. Separation based on reversible interaction between target protein and a specific ligand.
- AC requires:
- Beads matrix e.g. agarose in column
- Solution containing substance that needs to be isolated
- Ligand: a molecule that specifically binds to the protein of interest
- A wash to elute non-bound substances
- Final wash containing competitive ligand
What are the steps involved in affinity chromatography?
- A ligand attached to spacer arms. This is then attached to an inert support. The ligand is specific to the protein
- The wanted proteins attach to the ligand. The unwanted ones pass through the column.
- The column is washed with a buffer containing competitive ligand. This ligand will compete with black ligand – which one has a stronger affinity for the protein? The purple one will have a stronger affinity- the protein will bind to competitive ligand and comes out as eluent.
- The ligand and protein is separated by dialysis
- The column matrix is made of inert support with arms and ligands attached to it.
- You can change the pH, ionic strength and/or temperature so that the protein-ligand complex is no longer stable.
What is two dimensional electrophoresis?
- Involves Isoelectric focusing (IEF) and SDS-PAG.
- more than 1,000 different proteins from E. coli can be resolved using this technique
- A protein passes through the gel in the column. Every protein has an Isoelectric point (no net charge). When it reaches its isoelectric point the protein stops. Down the column the isolectric points decreases – the column is made of different pHs so when the protein reaches a pH equal to its isoelectric point the protein stops.
- The protein is added to a different separation material called SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). When the column of gel is placed here, the proteins will be separated based on molecular mass downwards and decreasing isoelectric point across.
- There will be standard so we can tell which protein is at which isoelectric point.
How is purification able to happen during two-dimensional electrophoresis?
- You can also purify just using first part of electrophoresis (SDS-page)
- You make a gel using polyacrylamide gel and put it into two plates creating a well and let in solidify.
- You place sample in the wells and pass electricity through it. The protein will separate based on molecular weight – heavier ones go through slower.
