Western Blotting

Identification of a specific protein in a complex mixture of proteins can be accomplished by a technique known as Western blotting, named for its similarity to Southern blotting,

Well coated with anti-cytokine

S ) Secretor

Non-secretor

Incubate at 37°C

Well coated with anti-cytokine

S ) Secretor

Non-secretor

Incubate at 37°C

Discard cells Wash plate

Discard cells Wash plate

Add enzyme-linked anti-cytokine antibody

Side view E = enzyme CS = chromogeni substrate CP = colored product

Add enzyme-linked anti-cytokine antibody

Side view E = enzyme CS = chromogeni substrate CP = colored product

Elispot Antibody Secreting Cells

Top view

In the ELISPOT assay, a well is coated with antibody against the antigen of interest, a cytokine in this example, and then a suspension of a cell population thought to contain some members synthesizing and secreting the cytokine are layered onto the bottom of the well and incubated. Most of the cytokine molecules secreted by a particular cell react with nearby well-bound antibodies. After the incubation period, the well is washed and an enzyme-labeled anti-cytokine antibody is added. After washing away unbound antibody, a chromogenic substrate that forms an insoluble colored product is added. The colored product (purple) precipitates and forms a spot only on the areas of the well where cytokine-secreting cells had been deposited. By counting the number of colored spots, it is possible to determine how many cytokine-secreting cells were present in the added cell suspension.

Top view

FIGURE 6-11

In the ELISPOT assay, a well is coated with antibody against the antigen of interest, a cytokine in this example, and then a suspension of a cell population thought to contain some members synthesizing and secreting the cytokine are layered onto the bottom of the well and incubated. Most of the cytokine molecules secreted by a particular cell react with nearby well-bound antibodies. After the incubation period, the well is washed and an enzyme-labeled anti-cytokine antibody is added. After washing away unbound antibody, a chromogenic substrate that forms an insoluble colored product is added. The colored product (purple) precipitates and forms a spot only on the areas of the well where cytokine-secreting cells had been deposited. By counting the number of colored spots, it is possible to determine how many cytokine-secreting cells were present in the added cell suspension.

(a) Add SDS-treated protein mixture to well of gel

(b) Electrophorese in

SDS-polyacrylamide gel r

Protein antigens denatured in SDS

Direction of migration

(b) Electrophorese in

SDS-polyacrylamide gel

(c) Remove gel and perform electrotransfer

Electric current

(c) Remove gel and perform electrotransfer

Electric current

Electrotransfer Dna

Porous membrane sheet

Porous membrane sheet

(d) Bind antigen of interest with enzyme-linked antibodies

(d) Bind antigen of interest with enzyme-linked antibodies

(e) Add substrate to activate color reaction

FIGURE 6-12

In Western blotting, a protein mixture is (a) treated with SDS, a strong denaturing detergent, (b) then separated by electrophoresis in an SDS polyacrylamide gel (SDS-PAGE) which separates the components according to their molecular weight; lower molecular weight components migrate farther than higher molecular weight ones. (c) The gel is removed from the apparatus and applied to a protein-binding sheet of nitrocellulose or nylon and the proteins in the gel are transferred to the sheet by the passage of an electric current. (d) Addition of enzyme-linked antibodies detects the antigen of interest, and (e) the position of the antibodies is visualized by means of an ELISA reaction that generates a highly colored insoluble product that is deposited at the site of the reaction. Alternatively, a chemiluminescent ELISA can be used to generate light that is readily detected by exposure of the blot to a piece of photographic film.

which detects DNA fragments, and Northern blotting, which detects mRNAs. In Western blotting, a protein mixture is electrophoretically separated on an SDS-polyacrylamide gel (SDS-PAGE), a slab gel infused with sodium dodecyl sulfate (SDS), a dissociating agent (Figure 6-12). The protein bands are transferred to a nylon membrane by electrophoresis and the individual protein bands are identified by flooding the nitrocellulose membrane with radiolabeled or enzyme-linked polyclonal or monoclonal antibody specific for the protein of interest. The Ag-Ab complexes that form on the band containing the protein recognized by the antibody can be visualized in a variety of ways. If the protein of interest was bound by a radioactive antibody, its position on the blot can be determined by exposing the membrane to a sheet of x-ray film, a procedure called autoradiography. However, the most generally used detection procedures employ enzyme-linked antibodies against the protein. After binding of the enzyme-antibody conjugate, addition of a chromogenic substrate that produces a highly colored and insoluble product causes the appearance of a colored band at the site of the target antigen. The site of the protein of interest can be determined with much higher sensitivity if a chemiluminescent compound along with suitable enhancing agents is used to produce light at the antigen site.

Western blotting can also identify a specific antibody in a mixture. In this case, known antigens of well-defined molecular weight are separated by SDS-PAGE and blotted onto nitrocellulose. The separated bands of known antigens are then probed with the sample suspected of containing antibody specific for one or more of these antigens. Reaction of an antibody with a band is detected by using either radiolabeled or enzyme-linked secondary antibody that is specific for the species of the antibodies in the test sample. The most widely used application of this procedure is in confirmatory testing for HIV, where Western blotting is used to determine whether the patient has antibodies that react with one or more viral proteins.

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