Ion exchange chromatography: Difference between revisions
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Ion-exchange [[Chromatography|chromatography]] separates [[Proteins|proteins]] according to their net charge. The stationary phase of the column will have a specific charge. | Ion-exchange [[Chromatography|chromatography]] separates [[Proteins|proteins]] and/or molecules according to their net charge. The stationary phase of the column will have a specific charge which is created by ion-exchange resins which come in two forms: polyanions (negatively charged) and polycations (positively charged). | ||
For example | For example, [[Proteins|proteins]] ([[Anions|anions]]) with a net negative charge will bind to a positively charged [[Diethylaminoethylcellulose|diethylaminoethylcellulose]] (DEAE-cellulose) column. The negative proteins bound to the column can be eluted by adding a negatively charged [[Buffer|buffer]] which will compete with the negative groups on the [[Protein|protein]] for binding to the column. Proteins that have lower charge density will be eluted first from the column<ref>Berg J., Tymoczko J and Stryer L. (2007) Biochemistry, 6th edition, New York: WH Freeman</ref>. | ||
A [[Protein|protein]] with a net positive charge (at a pH value of 7) will bind to a negatively charged column. The column will usually contain carboxylate groups ([[Carboxymethylcellulose|carboxymethylcellulose]] column). The bound protein will be eluted by increasing the concentration of a salt in an eluting [[Buffer|buffer]] (E.g. NaCl). The positive ions in the salt will contend with the positively charged groups on the protein for binding to the column<ref>Berg J., Tymoczko J, Gatto G Jr. and Stryer L. (2015) Biochemistry, 8th edition, New York: W.H. Freeman. Page 69.</ref>. | |||
< | The stationary phase is an insoluble matrix which will either carry a positive or negative charge. Whilst diethylaminoethylcellulose ([[DEAE-cellulose|DEAE-cellulose]]) is positively charged, an negatively charged matrix includes [[Carboxymethylcellulose|carboxymethylcellulose]] (CM-cellulose)<ref>Alberts et al. (2008) Molecular Biology Of The Cell, 5th Edition. Chapter 8, page 514.</ref><ref>Matthews, Van Holde, Ahern (2000) Biochemistry; Third Edition; P151</ref>. | ||
Ion Exchange Chromatography Video Link: https://www.youtube.com/watch?v=q3fMqgT1do8 | |||
=== References === | === References === | ||
<references /> | <references /><br> | ||
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Latest revision as of 09:01, 21 October 2018
Ion-exchange chromatography separates proteins and/or molecules according to their net charge. The stationary phase of the column will have a specific charge which is created by ion-exchange resins which come in two forms: polyanions (negatively charged) and polycations (positively charged).
For example, proteins (anions) with a net negative charge will bind to a positively charged diethylaminoethylcellulose (DEAE-cellulose) column. The negative proteins bound to the column can be eluted by adding a negatively charged buffer which will compete with the negative groups on the protein for binding to the column. Proteins that have lower charge density will be eluted first from the column[1].
A protein with a net positive charge (at a pH value of 7) will bind to a negatively charged column. The column will usually contain carboxylate groups (carboxymethylcellulose column). The bound protein will be eluted by increasing the concentration of a salt in an eluting buffer (E.g. NaCl). The positive ions in the salt will contend with the positively charged groups on the protein for binding to the column[2].
The stationary phase is an insoluble matrix which will either carry a positive or negative charge. Whilst diethylaminoethylcellulose (DEAE-cellulose) is positively charged, an negatively charged matrix includes carboxymethylcellulose (CM-cellulose)[3][4].
Ion Exchange Chromatography Video Link: https://www.youtube.com/watch?v=q3fMqgT1do8
References
- ↑ Berg J., Tymoczko J and Stryer L. (2007) Biochemistry, 6th edition, New York: WH Freeman
- ↑ Berg J., Tymoczko J, Gatto G Jr. and Stryer L. (2015) Biochemistry, 8th edition, New York: W.H. Freeman. Page 69.
- ↑ Alberts et al. (2008) Molecular Biology Of The Cell, 5th Edition. Chapter 8, page 514.
- ↑ Matthews, Van Holde, Ahern (2000) Biochemistry; Third Edition; P151