Key Concepts
- Electrophoresis is a separation technique based on the mobility of ions in an electric field.
- Electrophoresis can be used to separate mixtures of amino acids or fragments of DNA.
- The mobility of ions in an electric field depends on:
- charge
Positively charged ions moved towards the negatively charged electrode.
Negatively charged ions moved towards the positively charged electrode.
Neutral particles will remain stationary.
- mass
Ions with the least mass will move further than ions with greater mass.
- DNA fragments are negatively charged due to the negatively charged phosphate groups making up the backbone of the molecule.
- Amino acids can be neutral, acidic, or basic, depending on the pH of the solution.
Separating DNA Fragments
DNA fragments are negatively charged due to the negatively charged phosphate groups making up the backbone of the molecule so all the fragments in the sample will migrate towards the positively charged electrode.
Electrophoresis can therefore be used to separate DNA fragments based primarily on the size of the fragment.
- Shorter fragments will have less mass and will travel further through the gel.
- Longer fragments will have greater mass and will travel less far through the gel.
The results of an electrophoresis experiment to separate DNA fragments are represented below:
| | sample loaded here | fragment
X | fragment
Y |
|
| Negative Electrode | | Positive Electrode
| |
Both fragments, X and Y, are negatively charged so they have migrated towards the positive electrode.
Fragment X is expected to have the greater molecular mass since it has travelled the least distance.
Fragment Y is expected to have the lower molecular mass since it has travelled the greatest distance.
Fragment X will be larger than fragment Y.
Separating Amino Acids
Glycine (symbol gly) is an amino acid.
| The isoelectric point for glycine is at pH 6.1. At pH 6.1 glycine has no overall charge. |
|
Glycine will remain stationary during electrophoresis. |
| At pH < 6.1 glycine has a positive charge. |
|
Glycine will migrate towards the negative electrode during electrophoresis. |
| At pH > 6.1 glycine has a negative charge. |
|
Glycine will migrate towards the positive electrode during electrophoresis. |
Cysteine (symbol cys) is also an amino acid.
| The isoelectric point for cysteine is at pH 5.0. At pH 5.0 cysteine has no overall charge. |
|
Cysteine will remain stationary during electrophoresis. |
| At pH <5.0 cysteine has a positive charge. |
| |
CH2-SH |
+H3N -
|
|
C - COOH
|
H |
|
Cysteine will migrate towards the negative electrode during electrophoresis. |
| At pH > 5.0 cysteine has a negative charge. |
|
Cysteine will migrate towards the positive electrode during electrophoresis. |
If a mixture containing both glycine and cysteine is buffered at pH 5.5 and then undergoes electrophoresis, the results of the experiment could be respresented as:
|
glycine
(positive charge) |
sample loaded here |
cysteine
(negative charge) |
|
| Negative Electrode |
|
Positive Electrode
| |
At pH 5.5 glycine is positively charged so it migrates towards the negative electrode.
At pH 5.5 cysteine is negatively charged so it migrates towards the positive electrode.
If a mixture containing both glycine and cysteine is buffered at pH 7.0 and then undergoes electrophoresis, the results of the experiment could be represented as:
|
| sample loaded here |
cysteine
(negative charge) |
glycine
(negative charge) |
|
| Negative Electrode |
|
Positive Electrode
| |
At pH 7.0 both glycine and cysteine carry a negative charge so both will migrate towards the positive electrode.
Glycine has a slightly lower molecular mass than cysteine and could be expected to travel a little bit further.
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