Assaad A.

Eid
ae49@aub.edu.lb
Room: 1-51
 Kinase Assay

A protein kinase is a kinase enzyme that modifies other proteins by chemically

adding phosphate groups to them (phosphorylation). Phosphorylation results in a
functional change of the target protein by changing enzyme activity, cellular location,
or association with other proteins. Up to 30% of all human proteins may be modified
by kinase activity, and kinases are known to regulate the majority of cellular
pathways, especially those involved in signal transduction. The chemical activity of a
kinase involves transferring a phosphate group from a nucleoside triphosphate
(usually ATP) and covalently attaching it to one of three amino acids that have a free
hydroxyl group. Most kinases act on both serine and threonine, other act on
tyrosine. There are also protein kinases that phosphorylate other amino acids, including
his5dine kinases that phosphorylate his5dine residue.
 IMMUNOPRECIPITATION (IP) PROTOCOL
Whole tissue
Media & supplements, homogenate Antibody production/labeling,
filtration, cell lysis Purification kits,
protein extraction kits Antibody
kits, Microton Devices,
Cell Lysate

Immunoprecipitation Catch and Release IP kits, Protein A/

G agarose, Chip kits

Western bloting Kinase Assay

A method that enables the purification of a protein.

An antibody for the protein of interest is incubated with a cell extract so that
the antibody will bind the protein in solution.

The antibody/antigen complex will then be pulled out of the sample using
protein A/G-coupled agarose beads.

This physically isolates the protein of interest from the rest of the sample.

The sample can then be separated by SDS-PAGE for Western blot analysis
or for kinase Assay.
IMMUNOPRECIPITATION (IP) PROTOCOL

1. Lysis buffers and other reagents

2. Preparation of lysates
3. Pre-clearing the lysates
4. Immunoprecipitation
5. Choosing the correct beads
 Lysis buffers

The ideal lysis buffer will leave proteins in their native conformation,
minimizing denaturation of antibody binding sites while at the same time
releasing adequate amounts of protein from the sample for subsequent
analysis.

Nonionic detergents such as NP-40 and Triton X-100 are less harsh than
ionic detergents such as SDS and sodium deoxycholate.

Other variables that can affect the success of IP include salt concentration,
divalent cation concentration, and pH.
 Pre-clearing the lysates

Pre-clearing the lysate can help reduce non-specific binding of proteins to

agarose or sepharose beads.

The end result will be a lowering of background and an improved signal-to-noise

ratio.

However, if the final detection of the protein is by immunoblotting, pre-clearing

may not be necessary, unless a contaminating protein is interfering with
visualization of the protein of interest.
 Choosing the correct beads

The basis for antibody affinity purification is the high affinity and specificity of
protein G and protein A for the Fc region of IgG from a variety of species.

Protein G and protein A have been immobilized to several different matrices

resulting in an excellent means of isolating IgG subclasses from ascites, cell
culture supernatants, and serum.

Protein G and protein A are bacterial proteins from Group G Streptococci and
Staphylococcus aureus, respectively. When coupled to Sepharose base
matrices, protein G and protein A create useful, easy-to-use media for routine
purification of antibodies.
 Choosing the correct beads

Protein G and Protein A differ in their binding strength to immunoglobulins

from different species and subclasses.
 in vitro kinase assay

Cells that express the kinase you want to study are lysed with detergent to produce crude cell
lysate in a tube.

An antibody specific to the kinase you want to study is added to the cell lysate. The antibody
will bind to your target kinase.

The mixture is incubated with large agarose beads that are coated with Protein A or G. Protein
A and protein G bind antibodies, so incubating your cell lysate-antibody mixture with the coated
beads will lead to your kinase being specifically concentrated onto the surface of the beads via
the interactions between the kinase and its antibody and the antibody and the Protein A beads.

The tube is centrifuged, bringing all the kinase-coated beads to the bottom of the tube. The
bead pellet is then incubated with a target substrate (i.e. the protein you think your kinase will
phosphorylate) as well as radiolabeled ATP. The gamma phosphate (and only that phosphate)
of the ATP is radioactive P-32. If the kinase-substrate match is correct, the kinase will transfer
the radioactive phosphate from ATP onto the substrate.

After the incubation, a sample is taken from the liquid portion of the tube and run on an SDS-
PAGE gel. This sample contains the substrate protein.

The gel is then dried and examined by autoradiography. Any radioactive samples on the gel can
be detected. If the substrate was phosphorylated by the kinase, it will show up on the
autoradiogram. If the kinase was inactive or was unable to phosphorylate the chosen substrate,
the gel will appear blank.
Kinase Assay
Assay of protein kinases using non-radiolabeled
ATP
Step 1: Selective IP of Akt using Immobilized Akt Antibody.
a) Add immobilized Antibody.
b) IP Kinase from cell extracts using Immobilized Akt Antibody.

Step 2: Incubate IP pellets in Kinase Buffer containing Recombinant fusion protein and cold ATP.

Step 3: Detect Recombinant fusion protein phosphorylation using phospho specific antibodies by
Western blotting and chemiluminescent detection.