Monday, 11 August 2008

Techniques in Molecular Biology: Western Blotting

Today I thought I'd go into a little detail on one of the most common experimental techniques used in molecular biology - one that I've done far too many times this summer - the western blot.

In general, western blots are a technique that is used to determine the presence of a particular protein from a biological sample. For example, let's say that you've introduced a plasmid carrying the gene for alcohol dehydrogenase into E.coli and you want to check to see if the bacteria are actively transcribing and translating the gene. An easy way to check this is with a western blot. Westerns are also a useful diagnostic tecnhique in medical labs: westerns can tell you whether a protein is the wrong size, or if a particular protein from a virus or bacterium is present in a sample from a patient.

The theory behind Western Blotting is rather simple. First, you need a sample of proteins. These can be obtained in a variety of ways but they way that I'm most familiar with is through the use of sonication. A culture of cells is grown which (supposedly) express the protein you're interested in. By subjecting these cells to very high frequency sound, the cells rupture and spill all of their contents. One can then put the sonicated sample into a centrifuge, so all of the cellular debris clumps together at the bottom of the test tube, and the proteins in the cell are left floating around in the supernatant.

Now that all the proteins have been isolated, you need to seperate all of them. This is done by using electrophoresis on a polyacrylamide gel. Polyacrylamide forms a mesh through which the proteins travel through when an electric current is applied. Bigger proteins travel though the gel slower than smaller proteins; this, then, allows us to seperate all of the proteins based on their size. At this stage, it is possible to use a protein stain to stain the gel. This will allow us to easily visualize the proteins on the gel. Unfortunately, unless you were working with a pure sample of your protein of interest, you'd see a large smear since the gell contains ALL the proteins from the sonicated cells. To determine the presence of your protein of interest, you'd need a way to specifically visualize your protein and not the rest. Western blotting allows us to do this with ease.

We can speficically visualize whichever protein we want, but polyacrylamide gels do not allow us to do this. Thus, the next step is to transfer the protins to a medium which we can use to probe for a specific protein. One commonly used material for this is nitrocellulose membrane. Nitrocellulose has a very useful property - pretty much any protein sticks to it like glue (this also makes it a little tricky to handle, because you dont want proteins from your hands sticking to it and messing up your results. By making a nitrocellulose-polyacrylamide gel sandwich, and applying an electric current, you can force the proteins in the gel to transfer to the nitrocellulose. The membrane should then contain the proteins seperated as they were on the gel.

Now you're ready to detect specific proteins. This can be accomplished by using antibodies against whichever protein you're interested in. Alot of companies sell antibodies for commonly used proteins. Alternatively, you can design your protein so that it contains a "tag" - a short sequence of amino acids - at one end of the protein which is absent in proteins naturally produced in cells. You can buy antibodies which recognize different tags, and thereby bind only to your protein of interest. These antibodies will bind directly to the proteins on the membrane. Next, secondary antibodies are applied. These will bind specifically to the first antibodies.

The secondary antibodies are the key to detection of your protein. Attached to the secondary antibodies is an enzyme. Which enzyme is attached is a matter of the method of detection used. There are multiple ways of detection, but the most commonly used methods are chemiluminescent detection and pigment production. Both methods work in a similar way; attached to the antibody is an enzyme which will convert a substrate into a product when applied to the membrane. In the case of pigment production, the product is a pigment which can be direcly visualized on the membrane. In the case of chemiluminescent detection, the conversion from substrate to product produces light, which can be detected on a piece of X-ray film. In both cases, the result is a dark band representing your protein of interest. Where there is a dark band, there is the secondary antibody/enzyme; where there is secondary antibody, there is primary antibody; and where there is primary antibody, there is your protein of interest.

The image to the right shows what this end result looks like. You can also run a protein ladder along side your sample, which will show the size of known proteins, so you can determine the size of your protein.The whole process of western blotting can take a few hours to complete (or all day if you're bad at it like me). There is, of course, alot more to western blotting than this; what I've presented is a very generalized idea of how western blotting works. Nevertheless, it is a reliable way to detect the presence of any protein you want in a sample, and has become a staple technique of molecular biology.


Anonymous said...

Thank you very much. It is easy to understand what is the processes of Western Blot in identifying the protein of interest. Any video in demonstrating it will be of great help to those who never do wet lab.

C.W.G.K said...

No problem! Glad I could help. I dont have any videos demonstrating how to do a Western, but just doing a search on YouTube for "Western Blot procedure" brings up a ton of results. There's sure to be some good ones on there!

pranamee said...

For Videos you may want to check this one This company has many such protocol tutorials for different techniques.

Good Luck!

pranamee said...

Like said, there is a real good tutorial put up on Youtube which is good for a beginner

Good luck!