Western Whats??

Western blots.

I do a lot of western blots. They can be a source of outbursts of happiness accompanied by triumphant dancing, or (and currently, most commonly for me) be the cause of crippling despair!

So what’s a western blot?

And how does it have such power to control a poor post-doc’s emotional well-being?

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It is actually a relatively simple method of finding out whether a particular protein is present in the sample you are investigating (for example, a type of cell line, a bit of tissue from the body or a blood sample). It can also tell you whether one sample has more or less of that particular protein than another sample, or the size of a protein you are interested in.

The reason for doing this is to determine whether cells of one type are working in the same manner as cells of another type. For example, a western blot could tell you whether  ‘disease’ cells have more or less of a particular protein, which may make cells act differently (see my previous blog I fiddle about with cells in a dish for a clearer explanation!).

Proteins are made up of different length chains of amino acids, which results in proteins of many different lengths. The longer a protein is, the larger its mass (as it consists of more amino acids), and shorter proteins are smaller. The mass of a protein is measured in ‘kilodaltons’ (kDa). Each sample that is to be investigated is made up of a mixture of hundreds of different proteins. Therefore, to find the one you are looking for, these proteins need to be separated by their size/mass. This is done by using an electric current to push the sample through a gel, which is essentially a kind of mesh.

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In this mesh, small proteins can pass through very quickly, whereas large proteins struggle to move through the gaps and therefore move slower. The proteins will now be separated by their size rather than being in one mixed mess. A protein ‘ladder’ is passed through at the same time, which has particular sized proteins stained with a dye so that you know what size proteins are present at what point down the gel mesh.

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Where’s the blot part?

Well, this isn’t actually the western blot – this is just the necessary preparation for a western blot. This stage is actually called ‘sodium dodecyl sulfate polyacrylamide gel electrophoresis (or SDS-PAGE)’ – but don’t worry about that.

The blot part comes next. So the proteins are trapped in a gel, but gel breaks easily and is very fragile, so the proteins are transferred over  – or blotted – to a fabric ‘membrane’ that is easier to work with. This blotting is achieved by using an electric current to force the movement of proteins out of the mesh onto the membrane, which consists of lots of little pores that trap the proteins and lock them into place.

Once proteins are locked into place on the membrane, antibodies can be used to pick out the specific protein that you are interested in. Each antibody is designed to be specific to a particular protein, and theoretically should only attach to that protein and that protein alone (although the reality is often different!). When the antibody is attached, there are several methods by which it can be detected with specialised equipment. When it is detected, a ‘band’ of protein is made visible on the membrane. The size of the detected band can then be estimated by comparing it to the ladder that has dyed proteins of already known sizes.

If more of the protein you are looking for is in your sample, the band will appear fatter (and thinner if there is less of that protein). If there have been changes to the protein that might affect its size, then the band will appear above (if larger) or below (if smaller) where it is normally found on the blot.

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(A finished western blot – the ladder is on the left, the band of protein is present in the middle of the membrane. Each blob is a different sample, and each sample has a different size blob/band, suggesting different amounts of that protein between them)

The happiness comes from whenever a blot works (and when the results are consistent with what you predicted!). The disappointment stems from the many times they don’t work…this can be due to many variables such as broken equipment, non-specific antibodies, contamination, poor detection, out of date chemicals, bad blotting, old samples, and most recently in our lab, dodgy water! Sometimes they might not work for any other reason than just because the western blot gods are angry that day.

One more thing…Why are they called ‘western’ blots?

Truthfully, there is no real reason. It is a hilarious biochemical in-joke. There was already a similar method of detecting DNA called a ‘Southern blot’ (named after its inventor Prof. Southern). A method of detecting RNA (the single-stranded version of DNA) was subsequently named as ‘Northern blot,’ so it was decided that the equivalent detection of proteins would carry on the theme. An ‘Eastern blot’ for the detection of protein modifications also exists.

 

Feel free to comment and let me know if this made sense, or if there is anything I’ve failed to explain clearly, or if there are more details you would like to know!

 

The Biocheminist

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8 responses to “Western Whats??”

  1. Roger says :

    Hi Biocheminist,

    I wonder if you could answer a few questions regarding your Western Blot post.

    When you talk about attaching antibodies to proteins, do you help the
    antibody to bind to the protein using a potentiator? If so what do you use?

    Where do you obtain the antibodies for the experiment? From doners?

    And finally, what specialised equipment do you use to detect the protein with the attached antibody?

    P.S. Sounds like your lab could do with a new DI water filtering system !

    U Roger

    • thebiocheminist says :

      Antibodies are proteins themselves – the chain of amino acids that makes up a protein also folds in on itself to form a 3D structure. The 3D shape of an antibody ‘fits’ the complementary shape of the protein that it detects, which means that they can bind together. Whereas the antibody shouldn’t bind to proteins that it doesn’t fit (ideally)! With good and specific antibodies, attachment is easy so no potentiators are required. In fact the antibody is mixed with a ‘blocking’ solution when applied to the membrane – this solution contains generic proteins that bind to all sorts of things, and so ‘blocks’ any non-specific protein on the membrane that your antibody might accidentally bind to. Commonly, this blocking solution is skimmed milk powder!

      Thousands of antibodies are commercially made and are just bought into the lab. These, and in-house made antibodies are created in rabbits, mice or cell lines. The target protein is introduced to the animal, and their immune system creates antibodies against it in the same way it would in response to an infection. The antibodies can then be purified from the blood. In cell cultures, the cells are genetically altered to produce and excrete the antibody, and it can then be purified from the culture.

      There are a few different detection methods to see the protein. One of the most common is by fluorescence; the antibody that binds to your protein is called the ‘primary’ antibody and can not be detected by itself. So then a ‘secondary’ antibody is added which includes a fluorescent tag, which binds to the primary antibody. The fluorescent signal can then be seen in a machine that can illuminate the membrane at the correct light wavelength.

      The other method is called enchanced chemiluminescence (ECL). The primary antibody attaches in the same way, and a secondary antibody is also added, but instead of a fluorescent tag, this has a tag that emits light when an additional solution is added, causing a luminescent reaction. The light can be captured on photographic film and is exposed and developed in the same way as in traditional photography, or can be seen in a digital reader.

      The filter got changed on our water purification system, but everyone is still very suspicious and have stuck with using the super-pure stuff!

      Hope that helps!

      • Roger says :

        Thanks for your reply Biocheminist,

        Sounds like you do have problems with antibodies binding to non-specific proteins. Is this an annoying problem or one that you can live with? How much does it affect the experiment?

      • thebiocheminist says :

        The short answer is: it depends! A good antibody won’t have that problem. If an antibody does bind to other things, then how many other things and at what size determines whether it’s a protein. So if your antibody only picks up one other band, and the size is vastly different to the one you are interested in, then you can get by just ignoring it. If you’re picking up hundreds of bands, or one that is very similar to the one you’re interested in, then you’ll need a new antibody! Ideally, antibodies should be validated – this can be done by ‘knocking down’ the gene that codes for the protein you are interested in (so that there is none of your protein), then repeating the western blot. If the antibody is specific, you won’t see any bands anymore (because there is none of that protein left), and anything else that still shows up you can be confident that it’s crap!

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