PCB 4233L Quiz 2: Labs 4-6

Procedure: spin column anion exchange chromatography

- The sample will be added to the column and spun. - Once it is applied, those proteins that are negatively charged will bind to the positively charged resin beads. - Gamma immunoglobulins are slightly positively charged at pH 8 and thus do not bind to the column. - Contaminating proteins, which have a negative charge, will bind to the column. - We expect our immunoglobulins to flow through the column and they will be found in the initial flow through. - The column is then washed with additional buffer to ensure that all neutral and positively charged proteins that have no affinity towards the matrix are removed. - The negatively charged particles elute from the column when the high salt buffer is added to the column. - The chloride ions begin to compete with the proteins for access to the positively charged matrix as the concentration of salt increases, displacing the negatively charged proteins and allowing them to be collected in the flow-through.

Procedure: Production of Fab and F(ab')2 fragments

- digest IgG with papain and pepsin - antibody fragments will need to be purified since there will be unwanted fragments present after the digest. - Papain will digest IgG into 2 Fab fragments and 1 Fc fragment. This enzyme cleaves IgG just above the hinge region, thus generating 3 fragments of equal size. - Pepsin will digest IgG into one F(ab')2 fragment and it will cleave Fc into several smaller peptides (pFc'). This enzyme cleaves the IgG just below the hinge region, which keeps the two Fab fragments together.

Notes

- sodium azide prevents bacteria from growing by being the terminal electron acceptor, thus stopping the ETC and preventing the generation of ATP. - There is a dead volume within the column matrix caused by the matrix having spaces between the beads of the Sepharose or agarose, which the buffer will reside due to capillary forces. This means there will be some unbound product left in the column that is not bound to the beads but cannot be eluted without additional buffer being added to the column.

Chromatography

A technique that is used to separate the components of a mixture by differential movement through a two-phase system, with one phase being a liquid and the other an insoluble matrix.

Summary of spin column anion exchange chromatography

Add sample --> Spin --> Protein bound to membrane --> Wash --> Spin --> Elute --> Purified protein

Staining the gel

After the samples have been run through the gel, the gel must be stained in order to visualize the protein bands. The stain used depends on what you want to visualize (protein, carbohydrate) and how much (or little) protein is present. We will use Coomassie Briliant Blue to stain the proteins.

2. Background on protein G and why it is used for affinity chromatography

Anion exchange chromatography uses electrostatic interactions. between the proteins in solution and side groups covalently attached to the column matrix. Affinity chromatography uses protein-protein interactions to bind and separate certain proteins from a mixture of other proteins. Protein G is a bacterial protein isolated from eh cell surface of group G streptococci. These bacteria avoid neutralization, complement activation, and phagocytosis by macrophages by binding serum immunoglobulins at their Fc domain, thus inhibiting their effector function with macrophage, helper T cells, and B cells The protein G is covalently bound to the column matrix and interacts specifically with gamma globulin using hydrogen bonding, electrostatic, hydrophobic, and van der Waals forces. this ensures that only whole immunoglobulins or Fc fragments of immunoglobulins adsorb to the protein G-bound column matrix. The desired products of our digest, Fab and F(ab')2 fragments will pass through

Antibodies

Antibodies are made after infection or vaccination to detect and eliminate invading pathogens and allergens by binding to the foreign agent. The bound antibody can have an effector function or neutralize the pathogen by preventing it from binding to the specific proteins on the target cell, thus inhibiting infection. Long-term immunity is developed when the foreign agent is internalized, processed, and expressed by antigen-presenting cells (APCs), which in turn stimulate antigen-specific helper T cells and specific B cells to secrete additional antibodies.

Gamma immunoglobulins (IgG)

Contain 3 globular domains which form a Y shape. 2 antigen-binding sites situated at the ends of the arms. The hinge region in the center of immunoglobulin allows the antigen binding arms to have some degree of flexibility

UNIT 4: ANION EXCHANGE CHROMATOGRAPHY EXPERIMENT

Goal: - separate charged proteins - obtain purified protein [IgG (gamma immunoglobulin) and negatively charged protein] - negatively charged proteins and contaminants (also negatively charged) will bind to the column - Gamma immunoglobulins are positively charged at pH 8 and do not bind to the column. We expect the IgGs to flow through the column and be found in the initial flow through - the column is washed with additional buffer to ensure all neutral and positively charged proteins are removed - the negatively charged proteins are captured in the column and eluted with a high salt buffer. The chloride ions compete with proteins for access to the positively charged matrix, displacing the negatively charged proteins and allowing them to be collected in the flow-through.

The spin column Q

Has a binding capacity of 4 mg of protein per 0.4 mL of volume, but needs to be equilibrated before a sample is added. The column is equilibrated by passing a low salt buffer through it. This ensures that the membrane is ready to adsorb negatively charged particles.

Neutrophils and Macrophages

Have Fc-gamma receptors on their surface, allowing them to bind to antibodies with exposed Fc domains. Phagocytosis cannot occur if the Fc domain is already bound by another protein, such as protein G, which can be found on certain genera of bacteria (group G streptococci)

Immunoglobulin

Made of 2 heavy chains and 2 light chains. Heavy chains overlap at the hinge region and bind to the C-terminus of the light chains. The two heavy chains are linked by a pair of disulfide bonds in the hinge region that are formed with the sulfhydryl (-SH) side chain groups present on cysteine residues. Oxidative conditions will allow two R-SH groups to react and form R-S-S-R bond that is very stable and covalent in nature. Each heavy chain is also covalently linked to a light chain with disulfide bonds resulting in a complex of 4 peptide chains bound to each other with 4 intrachain bonds.

Fab fragments

Not affected by Fc receptors on macrophages, B cells, T cells, neutrophils, and mast cells. Do not precipitate antigen. Monovalent (1 antigen-binding site). Easier to make and purify than F(ab')2. Less likely to be phagocytosed by macrophages. Smaller size = greater access to hard to reach antigens

F(ab')2 fragments

Not affected by Fc receptors on macrophages, B cells, T cells, neutrophils, and mast cells. Will precipitate antigen Divalent (2 antigen-binding sites)

Anion exchange chromatography

Performed using ammonium sulfate precipitated fraction of goat serum, which was later dialyzed. The anion exchange spin column uses membrane-adsorbent technology as a chromatographic matrix to fractionate charged proteins. The Pierce Strong Ion Exchange Spin Column Q is a strong anion exchanger. The membrane is positively charged and exchanges with negatively charged particles. In our case, when the pH is 8, our protein will have a net positive charge when the charge from each amino acid is summed.

2. Production of Fab and F(ab')2 fragments

Prepare your IgG fractions for cleavage by papain or pepsin Equilibrate papain/pepsin for digestion Digest your IgG with papain or pepsin

1. Papain and pepsin digests need to be purified

Previously, you obtained gamma immunoglobulins from serum samples by ammonium sulfate precipitation, dialysis, and subsequent purification by anion exchange chromatography. Gamma immunoglobulins were digested with papain and pepsin. The products of these digests are Fab and Fc fragments or F(ab')2 and various short peptide fragments, respectively. These products need to be purified to remove any undigested immunoglobulins and unwanted products prior to their use in immunochemical and immunological techniques, which could interfere with your results and generate misleading data.

1. Anion Exchange Chromatography

Purify your salt-precipitated protein by anion exchange

Purification conditions

Sample: Ammonium sulfate precipitated fraction of serum IgGs that was desalted by dialysis Buffer A: 25 mM Tris*HCl buffer, pH 8.0

1. Introduction to SDS-PAGE

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) A technique used to separate proteins much like DNA fragments through an agarose gel. SDS is an anionic detergent that is used to coat proteins with a uniform charge that is proportional to the molecular weight and independent of the amino acid sequence. Acrylamide is a substance that polymerized into a gel-like matrix. The negatively charged protein-SDS complexes will migrate through the acrylamide gel according to their size rather than by charge. A marker containing proteins with known molecular weights can be used to estimate the sizes of the sample components.

Gerald Edelman and Rodney Porter

determined the structure of antibodies based on their cleavage patterns by different proteolytic enzymes; used chemicals to break the disulfide bonds.

Antibody fragment applications

immunohistochemistry immunoassays, including in vitro diagnostic assays radioimmunolocalization for tumor detection and radiotherapy immunotargeting through use of fragment conjugates as immunotoxins study of Fc binding proteins and effector functions

Ion exchange chromatography

the chromatographic procedure in which cationic or anionic proteins in the mobile (liquid) phase are separated by electrostatic interactions with the stationary (solid) phase

Cation exchange

the process in which a cation (+ charge) in a liquid phase exchanges with another cation present as the counter ion of a negatively charged solid polymer (cation exchanger). a cation exchanger is an insoluble solid that has negatively charged radicals attached to it that can attract and hold cations that pass by within a moving solution if they are more attracted to the acid groups than the counter ion present (such as chlorine, carbonate, or phosphate ions). Carboxymethylcellulose (CM) is a common cation exchanger used to separate positively charged proteins

Anion exchange

the process in which an anion (- charge) in a liquid phase exchanges with another anion previously bound to a solid, positively charged phase, the latter being an anion exchanger. an anion exchanger is an insoluble solid with cation groups that can attract and hold anions that pass by in a moving solution in exchange for anions previously held. Diethylaminoethyl (DEAE) cellulose is a common anion exchanger used to separate negatively charged proteins.

3. Molecular interactions within the affinity column

The IgG and Fc fragments will bind to the protein G when the sample is applied to the column. The sample will percolate through the column matrix using gravity. A peristaltic pump is not used because we want to maximize the amount of binding between our unwanted products and the matrix. Increasing the amount of time the sample is in contact with the matrix, pre-equilibrating the column with neutral pH buffer, and making sure the column has enough bed volume will ensure that the eluate that flows through the column does not contain Fc-possessing proteins. After the sample has flowed through the column, a small amount of eluate is still present withi the matrix. This can be collected and the column stripped of its bound proteins by adding an acidic buffer to the column. The acidic buffer increases the number of protons in solution, which interfere with the protein-protein interactions that keep IgG and Fc fragments bound to the column. We add a small amount of 0.2 M glycine hydrochloride to the column and check the pH using litmus paper. The pH will remain constant until the glycine-HCl reaches the bottom of the column and is eluted. We can stop collecting hte sample from the column at this point because the unwatned products of the digest will be leaving the column. Our goal is to collect as much of our desired products as possible without collecting the proteins that possess an Fc domain.

Analysis

The SDS-PAGE will be performed ot analyze the proteins which are present in the salt precipitated sample, the supernatant sample, the original IgG sample used for the digests with pepsin and papain, and the affinity-purified antibody digestion samples. All samples will be analyzied by using non-reducing codntions but the IgG and IgG digest samples will also be analyzed under reducing conditions.

Gel

The gel used in SDS-PAGE is a polyacrylamide discontinuous gel. The discontinuous gel has two components. 1. stacking gel - to concentrate large volumes, increase resolution 2. separating gel - to separate the sample components continuous gels lack the stacking gel. A solution of acrylamide and bisacrylamide is induced to polymerize by the addition of the catalysts, ammonium persulfate, and TEMED. The gel is poured within a pair of glass plates and with a sample comb, which determines the number of samples the gel can hold. once the gel has polymerized, it is loaded into the gel running apparatus, which holds the plates and running buffer while the electric current is being applied.

Column chromatography

a form of partition, adsorption, ion exchange, or affinity chromatography in which one phase is liquid flowing down a column packed with the second phase, a solid; the dissolved substances form a partition between teh solid and liquid phases depending on the chemical and physical conditions of each phase