understanding Western Blot

[bobat]

I am sorry if my question should be common sense, I am a first year student.

 

I am trying to understand exactly what a Western Blot test determines. In my book it says that a protein has a "lock and key fit with its protein target" and is found by dipping the membrane in a solution with an antibody. In my mind this makes it sound like the test locates the protein by seeing if it fits with its antibody....it does not sound like the test just locates the antibody.

 

I recall that Western Blot tests are used when testing for Herpes and HIV but every site that I find describing the specific test for these two viruses says the western blot test does not locate the virus itself but only the antibody. From what i read in my book it makes it sound as if the western blot test finds the virus itself by seeing if it binds to its corresponding antibody. So now I am completely confused.

 

Can anyone who understands my confusion clear things up a little bit?

[Genecks]

Western blots are used to determine if a protein exists within a biological specimen. or vial, or solution, or something...

 

Western blots use an agent to bind to the protein of interest; if the protein of interest exists, then the binding agent will attach to it.

 

So, imagine having different proteins run through the gel. Then, you apply an radioactive labeled antibody to the gel. If bindings occur, you'll be able to see strands on X-ray paper.

 

Otherwise, no attachments mean no bands.

 

Lock and key is a pathetic way to say "molecular bonding."

There is a large specificity for certain molecules to bind to other certain molecules.

This might have to do with the charges or shapes of the molecules.

 

But in another sense, most molecules (well, the wobble hypothesis is different) have a lock-and-key fit: only one molecule can bind to another particular molecule.

 

I suggest taking some time to read the wiki: http://en.wikipedia.org/wiki/Western_blot

 

Read it and re-read it.

Click on the various blue links in order to know the terms involved.

It will take a little time to understand what is all involved.

 

p.s.

 

I think you can use the western blot to detect an antibody. I'm not sure if it's the same routine, though.

In general, when someone is talking about using a test to detect proteins, then usually you should be thinking "Western blot."

Edited January 17, 2010 by Genecks

[zule]

Antibodies are proteins, so they can be detected by other antibodies.

[Genecks]

I thought other tests were used for viruses, but let's say were looking at viruses and various proteins involved in relation to western blots.

 

We could discuss proteins that are part of the virus.

In that sense, we would use a labeled binding agent (antibody, perhaps) and apply it to the gel sample to see if the protein that's part of that virus is there. Such a process could be done to see if a virus is present.

 

Furthermore, a test could be done to see if an antibody is present. Many antibodies attack viruses. Thus, a person would run the sample (possibly containing the antibody) through the gel, and he/she would attempt to attach a binding agent to an antibody. If attachment occurs, then this kind of information could tell you that a person indeed is making antibodies to fight off a virus.

 

Of this post, example one could tell you that a virus is present. In example two, it could tell you that antibodies are being made against a virus.

[CharonY]

Essentially the Western blot is a technique to immobilize proteins on a membrane, usually after applying a separation technique. The majority of Western blots utilize antibodies due to their specifiicity for detection as well as quantification. However any other visualization method is in theory applicable. It is a very universal method thus applied in many field of protein analyzes. It has become slightly less popular in cases when MS based methods are available, though.

As viruses are in relative low amounts in a body one rarely try to detect the virus or virus proteins directly but by using the fact that usually the hosts will raise antibodies against the virus. The latter is far easier to detect in e.g. blood samples.