Iso-electric focussing: Difference between revisions
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Iso-electric focussing ('''IEF''') is a molecular separation technique, often used to separate proteins. | Iso-electric focussing ('''IEF''') is a molecular separation technique, often used to separate proteins. | ||
'''IEF''' works on the basic principle of a proteins isoelectric point ('''pI'''). This is the pH at which a protein has an overall charge of zero. | '''IEF''' works on the basic principle of a [[proteins|proteins]] [[isoelectric point|isoelectric point]] ('''pI'''). This is the pH at which a protein has an overall charge of zero.<br> | ||
== Procedure == | |||
Firstly a gel is established with a '''pH''' gradient. This is acheived by the [[electrophoresis|electrophoresis]] of a mixture of [[polyampholytes|polyampholytes]] (small multi-charged polymers). | |||
You then add the sample in the middle of the gel and apply a voltage. This will involve a cathode at one end and an anode at the opposite end of the gel. | |||
The proteins will then start to move according to their charge. Positively charged proteins will move towards the [[cathode|cathode]], and negatively charged proteins will move towards the [[anode|anode]]. Once the proteins reach a point on the gel where the '''pH = pI''', the protein will stop moving. | |||
This separation technique is so precise, it can separate proteins with as little as one net charge differences <ref>Berg J., Tymoczko J., Stryer L., (2012) Biochemistry, 7th Edition, New York: W. H. Freeman and Company. pg 75. </ref> | |||
== Uses == | |||
<br> | When the proteins reach their '''pI''', they form bands, these can be removed and used in further experiments.<br> | ||
== References == | |||
<references /> | |||
Latest revision as of 19:07, 28 November 2011
Iso-electric focussing (IEF) is a molecular separation technique, often used to separate proteins.
IEF works on the basic principle of a proteins isoelectric point (pI). This is the pH at which a protein has an overall charge of zero.
Procedure
Firstly a gel is established with a pH gradient. This is acheived by the electrophoresis of a mixture of polyampholytes (small multi-charged polymers).
You then add the sample in the middle of the gel and apply a voltage. This will involve a cathode at one end and an anode at the opposite end of the gel.
The proteins will then start to move according to their charge. Positively charged proteins will move towards the cathode, and negatively charged proteins will move towards the anode. Once the proteins reach a point on the gel where the pH = pI, the protein will stop moving.
This separation technique is so precise, it can separate proteins with as little as one net charge differences [1]
Uses
When the proteins reach their pI, they form bands, these can be removed and used in further experiments.
References
- ↑ Berg J., Tymoczko J., Stryer L., (2012) Biochemistry, 7th Edition, New York: W. H. Freeman and Company. pg 75.