WSU Biochem 108 (Chromatography 1)

Mobile phase

A liquid (or a gas in gas chromatography) that is run through the stationary phase in chromatography.

Benefits of detecting a protein by function?

Determines if the protein is active and folded. Must be very specific.

Two kinds of resin used for affinity liquid chromatography

Free ligand Salt addition or altering the pH.

Dextran in LC

Glucose polymer (sephadex) Sugar polymer

Types of liquid chromatography (3)

HPLC (high-pressure LC) FPLC (fast protein LC) Conventional

What type of chromatography is used for RP?

HPLC or fast protein.

Elution gradient in HIC

High to low salt (instead of low to high in RP).

Analytical applications of SEC

Include determining the oligometric state of the protein and protein-protein interactions. 20kDa + 40kDa proteins will elute together at 60 kDa

Anion exchange chromatography

Negative proteins stick to positive beads, only positive proteins go through

TLC Mobile phase

Organic solvent.

Paper chromatography materials

Polar paper; hydrophobic solvent.

NTA (nitrilotriacetic acid) resin

Protein is eluted off with imidazole or with changing pH.

Western Blot general process

Protein is transferred from the gel to the membrane and then an antibody specific to the protein of interest is used for detection.

Description of the polar to non-polar mobile phase

Protein will elute off the hydrophobic surface and enter the mobile phase as the solvent becomes more and more non-polar.

What will cation-exchange chromatography capture?

Protons from the buffer will associate with the negatively charged resin and will be exchanged with positively charged residues in the protein.

Detection by size/sequence?

SDS-PAGE

How to use salt to elute a protein

Salt addition will weaken the electrostatic interaction between the ligand and the protein, and the protein will elute off.

Size-exclusion chromatography (SEC)

Separation based on size via gel filtration. Resin has pores of varying sizes. Larger molecules can't pass through the pores, and small ones can enter them.

V0 SEC

Solvent volume around the beads. It's usually~35% of Vt

Silicon in LC

Stands up to HPLC. Soluble pH > 8

Strong ion exchangers vs. weak exchangers

Strong: Fixed + or - charge Weak: Groups that titrate with pH

Stationary phase of PC

The paper that is handing from a glass rod (paper is parallel to the glass jar).

How to prevent retardation of the slope

Use KCl or NaCl to prevent the protein from interacting with the resin.

Key concepts of Ion-exchange LC (3)

Utilizes differences in sign and magnitude of the net charge on a protein at a given pH. Anion exchange vs. cation exchange. Strong vs. weak ion exchangers

How to detect if proteins of interest in LC

Absorbance. Proteins 214-280nm

pI < 7

Acidic protein meaning it contains a large number of aspartate and glutamate residues. At pH 7 these residues are negatively charged. You want a column positively charged (cationic resin = anionic exchanger).

Conventional LC method Advantages/Disadvantages

Ambient pressure where flow is due to gravity. Slower than the rest, allowing more diffusional spreading of the protein bands (much less resolution) but it's the cheapest. Can use large scale of proteins.

Anion vs. cation exchange

Anion-exchange column will have a positively charged resin and exchanges anions. Cation-exchange will have a negatively charged resin and exchanges cations.

Cation-exchange chromatography materials

Anionic resin. Column is anionic and is made up of a negatively-charged resin. Use with protein that have a high pI (lots of K and R)

General procedure of chromatography

Apply complex mixture in mobile phase to the stationary phase. Alter solvent conditions to change preference between phases Components become fractionated Separation down the length of the column.

Two classes of methods to search for elutant

Assay by size/sequence Assay by function

Goals of affinity liquid chromatography

Based on ligand-binding. Protein of interest is fused to other entities which can be used for purification using affinity chromatography.

Vt in SEC

Bed volume Total volume of resin and solvent

Fast protein liquid chromatography advantages and disadvantages

Can increase the capacity of the protein and still get pretty good resolution. Also, the resin does not have to be highly specialized making it cheaper.

Weak and strong ion exchange resins for cation-exchange chromatography

Carboxymethyl (CM) and phosphate for weak. Strong: methyl sulfonate (MonoS) Remember that these resins are positively charged.

Which ion exchange resin to use for acidic and basic proteins:

Cationic for the proteins with high pI (basic) and anionic for the proteins with low pI (acidic).

Anion exchange chromatography materials

Cationic resin. Use with proteins that have low pI (D, E).

Types of resin matrix (6)

Cellulose Dextran Agarose Acrylamide Poly-styrene Silica

Common ion exchange resin functionalities:

Charge affinity

Different protocols of liquid chromatography (4) and their corresponding resins

Charge: ion exchange chromatography Polarity: reverse phase, hydrophobic interaction chromatography. Binding: affinity chromatography Size: Gel filtration chromatography

Cellulose in LC

Cheap, but not durable Sugar polymer

CAT assay

Cloramphenicol acetyltransferase assay which is a reporter system using TLC protocol.

affinity liquid chromatography materials/set up

Column with resins that are linked to the ligand. Protein of interest will bind to the ligand. Wash off any unbound or loosely bound protein. Elute the bound protein by adding free ligand (may be expensive). Bound protein will bind to the free ligand, and elute off the column

Goal of polar paper chromatography

Detection of different pigments including pigments like carotenoids and chlorophyll. Fluorescence molecules can be separated and then visualized under UV light. Ninhydrin reacts with amino acids and will form bright purple spots.

Utility of SEC:

Determining native molecular weight Protein-protein interactions.

Dialysis utility

Dialysis allows exchange of buffer components across a semi-permeable membrane. Equilibrium dialysis can be used to determine binding affinity.

Poly-styrene in LC

Dowex Indestructible, but it denatures protein. Good for organic compounds and small peptides.

Detection by sequence?

ELISA or Western Blot Antibody-based detection. Recognizes either the primary sequence or a particular conformation of the protein of interest.

EMSA

Electrophoretic gel mobility assay: creates bands a piece of DNA that is radioactive we open the cell and the protein will bind to the DNA the gel. The gel is run and shows the band and then the band is identify to which protein it is associated with. bound probe and free probe meaning DNA Filter-binding with radioactive ligands.

RP chromatography general process

Elution gradient: polar to non-polar. Denatures proteins :( but excellent for peptides (protease digest). Hydrophobic proteins will bind to the stationary phase rather an interact with the polar solvent. Can move from polar to non-polar mobile phase by adding an organic solvent like acetonitrile or methanol. This will denature the protein and purify the peptides or for separating peptides in a protease digest (usually with HPLC or fast protein).

Detection of protein by function? (2)

Enzymatic assay Binding assays (EMSA)

General set up of LC

Equilibrate the resin introducing the correct buffer to the resin. A good buffer is a low salt with appropriate pH. This is the mobile phase. Load the sample (complex mixture) to the column. Wash the column in between sample loading to remove any proteins that did not bind to the column, Elute the protein off the column usually using a gradient (salt gradient, the column goes from low salt to high salt concentration).

Disadvantages/advantages of Affinity liquid chromatography

Expensive Require a lot of ligand. Powerful, because it's specific.

HPLC advantages and disadvantages

High pressure will speed up movement of the protein molecules down the column, reducing the diffusional spreading. High resolution. These systems have low capacity... cannot separate large volumes of protein or other macromolecules.

IMAC (Immobilized metal affinity chromatography)

His Tag formation to simulate affinity of his to metals: N terminus or C-terminus of the protein is fused to 6 histidine. His has a high affinity for metals (including nickel). If the resin has nickel appended to it, the protein will bind to the nickel.

Fusion proteins

His tag: Ni2+ column GST fusion (glutathione S-transferase) MBP fusion (maltose binding protein).

Description of HIC set up

Hydrophilic surface with hydrophobic groups attached to the surface. Ammonium sulfate typical salt used which enhances the hydrophobic effect. Strong binding at high salt concentrations between the hydrophobic patches on the protein surface and the hydrophobic groups on the resin. When salt concentration is being lowered, the proteins that have smaller hydrophobic patches in terms of surface area will elute first and the ones with larger hydrophobic patches elute later.

Relationship between MW and SEC elution:

I think this is the largely linear relationship discussed earlier.

What to worry about SEC (3)

If the protein interacts with the resin, there would be unintended retardation affecting the elution time or elution volume. Sample volume should be 1-2% of total volume allowing you to get enough resolution of the protein sample. Analytical applications

Goal of SEC

Isocratic elution. There is no gradient change in elution buffer. Larger proteins will elute before smaller ones. Larger proteins that cannot enter the pores will elute straight through the column. Smaller proteins entering the pores delay the emergence of the protein.

How do we know when to use cation or anion exchanger?

Know the pH of the buffer. pI of the protein.

What happens to non-target molecules in SEC

Large molecules that do not pass the exclusion limit will come out in the void volume. No separation.

Relationship between Ve and log(MW)

Largely linear negative slope. Standard curves can be generated in this relationship.

Exclusion limit SEC

Mass of the smallest molecule unable to enter the pores of resin.

Two phases of chromatography

Mobile and stationary

Why use HIC?

More gentle and non-denaturing as RPC Based on polarity of protein surface. Uses typical sugar resins (sepharose). Can purify active proteins.

Agarose in LC

Polymer of Gal, deoxy Gal (sepharose) From seaweed, cross-linking improves durability. Sugar polymer

Stationary phase

Porous solid material (matrix) which is held in a column.

Why DEAE for the anionic exchange?

Positive charge is a function of pH.

HPLC

Pressure of about 800 psi used to separate proteins. Stationary phase - polar Mobile phase - nonpolar

Difference between RP and HI chromatography

Salt gradient concentrations: In RP it's low to high, HI chromatography vice versa.

What if you have a large concentration of salt within the membrane

Salt will travel from inside to the outside the membrane until the salt concentrations on each side are equal.

Brief description of dialysis.

Semipermeable membrane (cellulose acetate) containing concentrated solution of the protein. Large molecules are proteins, DNA, or RNA. Buffer and salt molecules are smaller.

What is chromatography typically used for nowadays

Separating amino acids, peptides, sugars, nucleotides, and other small simple molecules.

Main goal of crhomatography

Separating proteins in to fractions based on properties including size, charge, or binding affinity.

Reverse phase (RP) chromatography goal

Separation based on hydrophobic properties (lack of polarity) Hydrophobic stationary phase = alkyl groups of C4 to C20.

Acrylamide in LC

Sephacryl Durable!

Thin layer chromatography solid phase

Silica on a glass plate

HPLC materials

Silicon durable enough to withstand pressure.

Fast protein liquid chromatography materials

Silicon durable enough to withstand some pressure but not as much as HPLC.

Dialysis set up (3)

Size separation across the membrane. Exchange buffers, remove salts. Different molecule-weight cut-off (MWCO) values for different membranes.

Other affinity examples: (6)

Small molecule ligands Antibodies Peptides Proteins Nucleic acids Pseudo-affinity dye resins

Strong ion exchange and weak ion exchange resin for Anion-exchange chromatography

Strong: Quaternary amine (MonoQ) Weak: Diethylaminoethyl (DEAE)

What is resin

Such that it can use the properties, chemical or physical, or the macromolecules to bring about separation.

What will anion-exchange chromatography capture?

The buffer has hydroxyl groups which will bind the positively charged groups on the resin. Negatively charged carboxylate groups (aspartate or glutamate) in proteins, they will exchange with the hydroxyl groups and bind to the column.

Mobile phase of PC

The solvent represents the mobile phase. Solvent will move up the paper with whatever non-polar materials you are separating that will spot quite high on the paper. Polar substances will not travel high up the paper.

pH > 7

Use anionic resin and cation-exchange chromatography

Fluorescence quenching

Used in TLC where inorganic phosphorus is mixed with the adsorbent before it is coated on the plate. Shine UV light on the developed chromatogram, the surface of the plate fluoresces with a characteristic color except in those places where UV-adsorbing solutes are situated. These quench the fluorescence and are visible as dark spots.

Basis of affinity chromatography and some examples (including fusion proteins)

Using proteins fused with tags and the cooperative resin in order to isolate these proteins. When nickel is added to a resin, you can add Histidine to the C or N terminus of proteins for them to be isolated. This is good for purifying proteins.

Difference between weak and strong exchangers:

Weak exchangers will depend on titration of pH whereas strong has a fixed charge. Strong exchangers include carboxymethyl and Quant. amides. Weak includes methyl and DEAE groups.

hydrophobic interaction chromatography

column chromatography that separates molecules based on their hydrophobicity (aversion to water molecules) Hydrophilic surface is decorated with hydrophobic substitutions.

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis Excellent for assessing purity. Not hard if the protein is over-expressed.