Immunoprecipitation and Western Blot

two methods commonly used in research to investigate protein-protein interactions, or to generally detect the presence of proteins in a sample

Immunoprecipitation and western blot

Immunoprecipitation and western blot take advantage of the ability of antibodies to

recognize epitopes in antigens

uses antibody to pull down proteins and co-interactors in a sample (usually a cell lysate)

Immunoprecipitation

The Fc region of IgGs have a high affinity to

bacterial protein A or G

How are antibody-antigen complexes are precipitated in Fc region of IgGs

addition of protein A or G-coated agarose bead, or magnetic beads

The presence of proteins after IP are then detected by

western blot

samples are ran in a denaturing gel, and transferred to a membrane. Proteins in the membrane are then probed with antibodies and visualized to see bands that signify the presence of the protein.

western blot

The fundamental goal of IP

purification and isolation of a specific protein using an antibody against that protein

The target protein might be

endogenous or recombinant

Goal of IP

Protein characterization

Protein-protein interactions

Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to

simplify subsequent purification

Typically, it is easier to optimize recombinant protein IP. Why?

antibodies against recombinant epitope tags are very strong and effective

Why is it much more difficult to optimize endogenous IPs?

Antibodies against endogenous proteins have extremely variable efficacy

A necessary step after immunoprecipitation is

verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots.

An important control is the use of a ________________________________ to verify pull down of the correct protein.

different antibody during the Western blot

The goal after purification may be characterization of the protein itself by

NMR, mass spectrometry, and in vitro assays, or analysis of the protein's interacting partners (i.e. protein, DNA, RNA)

After production of lysate from cells, there are two major steps in IP

pre-clearing and pull down

During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an

isotype control antibody

In pull down step, the target protein is pulled down using a

protein-specific antibody

TRUE OR FALSE

Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains.

TRUE

A key component of IP is __________________ that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein.

Protein A/G agarose beads

In IP, the constant domain identifies

type of antibody and dictates function in vivo

Usually, the constant domains of antibodies used for IP are

mouse, rat, or rabbit IgG

The antigen binding portion of the antibody recognizes

a specific epitope of a specific protein

the availability of the epitope depends on ______________ which is identifying it is an important factor to consider when choosing antibodies and conditions for IP.

protein folding

Why is pre-clearing step in IP necessary?

to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product.

second key component of IP after isotype and protein-specific antibody.

Bacterial antibody-binding proteins

Bacterial antibody-binding proteins function and examples

pull down antibody: protein complex

Example: Proteins A, G, and L

beads and spin columns are used for

smaller sample sizes

resins are used for

bulk purification

Besdies protein foldings, another factor to consider when choosing conditions for IP.

proteins have different binding affinities for different species and different constant domain subtypes

Process of IP

1. Lysates cells (chemicals or sound waves)

2. Pre-clearing

3. Pull down

In lysation, once the cell membrane is broken up, the hydrophobic phospholipids will associate together forming

small liposomes

used to identify the presence of specific proteins in electrophoretically separated samples

Western Blotting

Following separation by a technique known as __________________, western transfer is used to move proteins from a polyacrylamide gel onto a piece of membrane which traps the proteins in their respective locations.

sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE

After SDS-page, the membranes are probed with antibodies in a process called

immunoblotting

uses antibody-protein and antibody-antibody binding through specific recognition sites, providing the high specificity required for identifying a single protein.

immunoblotting

The detection of antibodies takes place using reporter systems which includes the use of

enzymes

Enzymes can be attached to the end of an antibody and react with substrates to produce changes in color or light. These signals can then be imaged and quantified using a process called

densitometry

There are 3 principal stages of WB that are essential for a quality outcome:

Electroblotting, Immunoblotting, and Detection

also known as the Western “transfer” and requires a transfer cassette for holding together the “sandwich” as well as an apparatus for transferring protein from an acrylimide gel to a thin membrane.

Electroblotting

The electroblotting sandwich consists of the

gel and a specialized membrane, sandwiched between two pieces of filter paper.

During western transfer, an electric field is used, to move the proteins through the gel, where they become trapped on a membrane due to

charged and hydrophobic interactions.

uses antibodies to “probe” the membrane for specific proteins

Immunoblotting

antibodies that recognize a single epitope and are the preferred antibody type used for immunoblotting due to their specificity.

Monoclonal antibodies

different antibodies that target many epitopes on the same antigen – or protein for which an antibody has specificity

polyclonal antibodies

Monoclonal antibodies that recognize a linear epitope are preferred. Why?

ensures the epitope can be found on a denatured, or linearized, protein. This is important because many antibodies only recognize conformational epitopes, which means that they recognize proteins in their native 3D state only.

In immunoblotting, this region is mainly utilized as the epitope for a secondary antibody – an antibody which recognizes the first antibody that has bound to the protein you’re trying to detect.

Fc region

In order to produce an observable signal, antibodies are often linked, through their Fc region, to a reporter enzyme, such as

alkaline phosphatase or horseradish peroxidase

reporter enzymes produce signals by

reacting with substrates to cause color changes or produce light changes.

technique used to measure the density of a protein band using image analysis software to calculate the density of each band.

densitometry

3 types of reporter enzymes that are commonly found on secondary antibodies are:

• a colorimetric western blot uses an enzyme-conjugated secondary antibody.  Results are detected using a chromogenic substrate (i.e., a substance capable of conversion into a pigment).

• a chemiluminescent western blot uses an enzyme-conjugated secondary antibody and a luminescent substrate. Results are detected using x-ray film and darkroom equipment.

• a fluorescent western blot uses a secondary antibody conjugated to a fluorophore.  Results are detected using a fluorescence imager.