Sunday, 31 May 2009

How does electrophoresis work?

In medicine, electrophoresis is a technique for separating (whole or fragmented) DNA, RNA or proteins on the basis of their sizes and electric charges.  Because the usual pattern in health may be disturbed by pathological conditions, the technique can therefore be used to diagnose such pathologies.

To conduct electrophoresis, you first have to place the sample of interest into a medium in which it can move about; this is usually a "gel" of polyacrylamide or agar.  This entire contraption is then subjected to an electric field.  This will cause molecules with a net positive charge to migrate towards the side with the cathode (negatively-charged terminal), and the molecules with a net negative charge to do the opposite (moving towards the positively-charged anode).

The important part here is that not all molecules will move an equal amount, let alone in the same direction, in the given time span.  Molecules with a stronger intrinsic electric charge will move faster than ones with only a low electric charge, and larger and asymmetrical molecules will move more slowly than their slimmer, streamlined counterparts.  (In the case of protein electrophoresis, however, the proteins are often uniformly coated with an electric charge so that only their sizes and shapes are relevant.)

After the specified amount of time has elapsed, the current is switched off and the mixture is stained with a dye that binds to proteins or nucleic acids.  The readings can now be made.  For instance, a sample of four different proteins might look like the image to the right, once separated by electrophoresis.

Now, if a patient was deficient in vSRC (don't worry for a second what that protein is), or had an abnormal vSRC, the pattern produced by electrophoresis would be different to that of a healthy control.

Similarly, once DNA has been chopped up into smaller fragments, fragments with abnormal or absent nucleic acids would also produce pathological staining patterns.

One final clinical example.  Multiple myeloma is a haematological neoplasm involving plasma cells which usually secrete abnormally large amounts of a particular immunoglobulin.  This 'spike' in the normal concentration of immunoglobulin can be readily appreciated with the help of electrophoresis.  The normal patient's serum protein electrophoresis would look something like this, in graphical form: 


The height of the blobs represents the "darkness" of the stain, which in turn reflects the amount of protein there.  The position of the blobs along the x-axis shows how far the various proteins migrated along the gel.  The large blob on the left is albumin (the most numerous of our serum proteins under normal conditions), and it is followed by blobs named α1, α2, β and γ. Each of the blobs with Greek letters contains several serum proteins that happen to have the similar electrophoretic properties and so are grouped together.  Have a look at the γ region, which is made up entirely of immunoglobulins (this is why they're sometimes called "gamma globulins"!).  That's what it should look like.

But we've already noted that patients with multiple myeloma often produce a huge amount of one type of immunoglobulin.  Concurrently, the formation of other normal immunoglobulins is suppressed.  Therefore electrophoresis of their serum protein will produce a picture something like this:


Serum protein electrophoresis (SPEP) is part of the routine workup of patients in whom you are suspecting multiple myeloma as a diagnosis.

5 comments:

  1. electrophoresississisissis

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  4. Yes electrophoresis technique is used to separate DNA, RNA or proteins according to their sizes and shape by applying electric charges. This technique is very useful in diagnosing many infectious diseases. It depends on the fact that when electric charge is applied on the DNA, they will shift toward the positive pole as they are considered as negatively charged.

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