protein chromatography - (Apr/16/2007 )
Can someone correct this for me:
Predict the elution sequence of the listed compounds on anion exchange, cation exchange and hydrophobic interaction chromatography.
Lys
Lys-Arg-Gly
Glu-Lys
Leu-Phe-Ala
Phosphorylated Tyr
Trp-Val-Phe
I tried this my best. My answers are:
On anion exchange, the resin is postive charged and will bind to neg charged proteins strongly, so the protein that is very neg charged binds strongest & elutes last on the column:
Elutes 1st: Trp-Val-Phe (very hydrophobic)
2nd: Leu-Phe-Ala (hydrophobic)
3rd: Lys-Arg-Gly (very postive charged)
4th: Lys (post charged)
5th: Glu-Lys (neutral)
6th: phosphorylated tyrosine (neg charged)
Cation exchange is just the opposite of anion exchange & protein that is most postively charged will elute last because cation exchange resin is neg charged:
Elutes 1st: Trp-Val-Phe (very hydrophobic)
2nd: Leu-Phe-Ala (hydrophobic)
3rd:
4th: Glu-Lys (neutral)
5th: Lys (post charged)
6th: Lys-Arg-Gly (very postive charged)
Hydrophobic interaction column will elute the most hydrophobic compound last
Elutes 1st: Lys-Arg-Gly (very post charged)
2nd: Lys (post charged)
3rd: Glu-Lys (neutral)
4th: phosphorylated tyrosine (neg charged)
5th: Leu-Phe-Ala (hydrophobic)
6th: Trp-Val-Phe (very hydrophobic)
Before you go any further, what pH are you working at? That will determine the charge, which will determine the order of elution. Also, if the peptide is uncharged, or the wrong charge for the column, it will elute en masse in the void, rather than be separated.
When you think about HIC, remember the elution is from high salt to low salt, so the most hydrophobic will elute first, not last. Again, if there are no hydrophobic residues, the peptide will pass through.
When you think about HIC, remember the elution is from high salt to low salt, so the most hydrophobic will elute first, not last. Again, if there are no hydrophobic residues, the peptide will pass through.
Aa addition to Swanny reply I think it will be reasonable you to determine pI of your peptides. You can calculate it easy.
Then chose pH of solution in what you imagine to dissolve them !
For ex: pI of your components are 4, 5, 6, 7, 8,
You choose pH of buffer to dissolve your peptides 6. At this pH peptides with pH 4 and 5 charged negative and peptides with pI 7 and 8 charged positively
So appling to anion exchange resin
peptides with pI 7 and 8 fall through the column without binding to resin
peptide with pI 4 will bind stronger to resin ( and eluted later) than pep with pI 5
So strategy apply to cation exchange resin
GOOD LUCK!
peptides with pH 4 and 5
To both,
This question does not give a pH or pI in either load or elution steps. So it does not work with pH or pI so much and thus can be ignored for this.
I think it is asking for a general treatment of the proteins and list them in order of elution.
In that case, are my answers correct?
This question does not give a pH or pI in either load or elution steps. So it does not work with pH or pI so much and thus can be ignored for this.
I think it is asking for a general treatment of the proteins and list them in order of elution.
In that case, are my answers correct?
Concerning IEC you can't explain elution order in terms of hydrofobicity but only in terms of charge. May be in this question suppose that you give this addition yourself if you understand the mechanism!
This question does not give a pH or pI in either load or elution steps. So it does not work with pH or pI so much and thus can be ignored for this.
I think it is asking for a general treatment of the proteins and list them in order of elution.
In that case, are my answers correct?
When you are talking about ion exchange, pH can never be ignored! If the question does not mention the pH of the system, you have a couple of choices: you can set your own pH for the thought experiment, and say what will happen at that pH, or you could be really general and say that at a given pH, if the peptide's pI is lower, it will behave in such-and-such a way, but if the pI is higher, then it will behave in so-and-so a way.
When you consider HIC, you only need to consider the hydrophobicity of the protein, because that is the determiner of how molecules will separate, or not separate.
It looks like the question has been written in a very general manner to allow you to give as general an answer as possible.