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38 Cards in this Set
- Front
- Back
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Ion Exchange Chromatography separates proteins based on ___
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charge
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The overall net charge of a protein is dependent upon the __ of the solution it is contained in
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pH
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A protein's ____ is the pH at which there is an overall net neutral charge on the molecule
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isoelectirc point (pl)
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A protein will not bind to a charged column if the pH is equal to __
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the protein's pl
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As pH increases above the pl, protons ___, leaving a net __ charge on the protein
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leave the side chains
negative |
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If the pH decreases below the pl, protons __ and cause an overall net __ charge
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associate with the protein
positive |
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A protein will bind to a column that is of ___ charge
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opposite
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Anion exchangers bind to __ charges
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negative
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Cation exchangers bing to __ charges
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positive
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The charges on the exchangers are balanced by the ___
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counterions
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___ is the counter ion for anion exchangers
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Cl
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___ is the counter ions for cation exchangers
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Na
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Anion exchange chromatography involves exchanging ___ charged anions by using beads of a ___ charge and a buffer with a pH __ than the pl of the protein to ensure it has a ___ charge
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(-)
(+) higher (-) |
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Cation exchange chromatogrpahy involves exchanging ___ charged cations by using beads that are ___ charged and buffer with a pH ___ than the pl of the protein of interest
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(+)
(-) lower (+) |
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The difference between the buffer pH and the protein's pl affects the rate at which _____
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the protein will travel through the column
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Proteins should be placed in a buffer of pH generally __ unit(s) or greater away from the pl to ensure that proteins will ____
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one
fully bind to the column |
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Prtoeins are eluted using ___
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NaCl
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Proteins in a buffer with a pH close to their pl will elute ___
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first
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A NaCl ____ is used to elute proteins one by one by slowly increasing the concentration of salt
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gradient
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To select the appropriate type of IEX matrix (cation or anion) one must understand ____
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protein charge
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The net charge of a protein is dependent on ___
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pH
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At its isoelectric point, the net charge of a protein is ___ and ___ binding will occur to the gel matrix
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zero
no |
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Choosing an appropriate IEX matrix also depends on the pH at which the protein is most ____
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stable
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To determine this a ___ test can be performed where the protein of interest is placed in various ____ and ___ and precipitation can be observed
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solubility
buffers and salts |
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If a protein is most stable at a pH above its pl it has a ___ charge
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negative
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If a protein is most stable at a pH below its pl it has a ___ charge
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positive
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the buffer should have a pH greater than ___ unit away from the pl of the protein to ensure: ___
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one
tight binding |
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Another way to get your proteins to attach to the column is to change the ___
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pH
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Anion exchange is not something you would do ___
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first
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The PI is the average of both ___
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pKas
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As pH increases :
Amino terminus: ___ --> ___ Carboxyl Terminus: ___ --> ___ |
NH3 +--> NH2
COOH ---> COO - |
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To select a column you must know the ___ of your protein
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isoelectric point
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To switch from an anion to cation exchange, you have to change the ___ and a ____ to get rid of the salt
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pH
buffer exchange |
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As pH decreases :
Amino terminus: ___ --> ___ Carboxyl Terminus: ___ --> ___ |
NH2 --> NH3+,
COO- --> COOH |
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If your pI is 5:
Cation exchanger pH< ___ Anion exchanger pH> ___ |
4
6 |
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Negatively charged columns are called ___
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CM or S
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Positively charged columns are called ___
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DEAE and Q
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Ion exchange is done in a range from ___ pH
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4-9
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