WesternBlots - ImmunoBlots                             

 

Principle of WesternBlot Procedure

 

In an Western blotting, bacterial or viral proteins are electrophoresed into a gel.

As the proteins migrate through the gel they are separated based upon size and charge. Characteristically, smaller proteins migrate through the gel faster than larger proteins. Sufficiently separated proteins in SDS-PAGE (sodium dodecyl sulphate polyacrilamid gel electrophoresis) can be transferred to a solid membrane for Westernblot analysis. For this procedure, an electric current is applied to the gel so that the separated proteins transfer through the gel and onto the membrane in the same pattern as they separate on the SDS-PAGE.

All sites on the membrane which do not contain blotted protein from the gel can then be non-specifically "blocked" so that antibody (serum) will not non-specifically bind to them, causing a false positive result. Often the membrane is cut into strips to facilitate testing of a large number of samples for antibodies directed against the blotted protein (antigen).

To detect the antigen blotted on the membrane, a primary antibody (serum) is added at an appropriate dilution and incubated with the membrane. If there are any antibodies present which are directed against one or more of the blotted antigens, those antibodies will bind to the protein (s) while other antibodies will be washed away at the end of the incubation. In order to detect the antibodies which have bound, anti-immunoglobulin antibodies coupled to a reporter group such as the enzyme alkaline phosphatase are added (e.g. Goat anti-human IgG- alkaline phosphatase). This anti-Ig-enzyme is commonly called a "second antibody" or "conjugate". Finally after excess second antibody is washed free of the blot, a substrate is added which will precipitate upon reaction with the conjugate resulting in a visible band where the primary antibody bound to the protein.

For example, for specific HIV diagnostic test, HIV infected cells are lysed, subjected to SDS-PAGE and blotted onto a membrane as described above. The membrane was blocked, cut into strips and incubated with the serum samples from each patient as indicated.

Below you can see an example of a Western Blot and how to interpret it.

Viral Proteins (HIV) and Band Pattern Interpretation

Viral Proteins - HIV, like any other virus, is composed of a number of different proteins. The Western Blot positive control lane contains proteins from patient sera as well as HIV proteins. HIV positivity can therefore only be confirmed by the presence of the following types of proteins:

gp160

viral envelope precursor (env)

gp120

viral envelope protein (env) binds to CD4

p24

viral core protein (gag)

p31

Reverse Transcriptase (pol)

Band Pattern Interpretation

In 1987 the Centre for Disease Control along with several other organizations established criteria for serologic interpretation of HIV Western blot tests. The criteria are listed below:

bullet

No bands present     -     Negative

bullet

Bands at either p31 OR p24 AND bands present at either gp160 OR gp120     -     Positive

bullet

Bands present, but pattern does not meet criteria for positivity     -     Indeterminate

Band pattern Interpretation

  1. Lane 1, HIV+ serum (positive control)
  2. Lane 2, HIV- serum (negative control)
  3. Lane A, Patient A negative result
  4. Lane B, Patient B indeterminate result
  5. Lane C, Patient C positive results

You can see also Helicobacter Pylori Westernblot Interpretation

 

FeedBack

 

E-mail: postmaster@westernblot.ru