oligonucleotide -protein conjugate

[zahra1634]

Hi,

I want to purify a conjugate of fluorescent tag-oligonucleotide-protein. I have started the simple HPLC using water/TFA/Acetonitrile solvent with 5-100% gradient . I was wondering if anyone could help me about which method HPLC, Ion-exchange,size exclusion, FPLC or UPLC would be better for that and which solvents .
Sorry if it is not kind of professional question as I have started HPLC analysis very recently.

Thannnnnkkkks

[unmgvar]

you are missing several key concepts of chromatography.
really in short:
IEX, SEC or RP are different types of chromatography chemistry types that can be used in order to achieve your separation either based on polarity for IEX, size exclusion for SEC or hydropobicity for reversed phase
FPLC (MPLC), HPLC, UHPLC are simply the use of those techniques in a certain range of pressure
for FPLC you will use columns with big particle sizes or mesh columns which give lower back pressures, generally as small as 10u. in many cases using a AKTA system with plastic self filled columns. pressures will hardly go above 50 bars.
for HPLC you will us columns with 10-3u particles sizes with generally pre-packed columns. pressures going from 50 to 200 bars
for UHPLC you will use columns of 3-1.5u particles sizes. pressures will range from 250 to 1000 bars
the normal HPLC is more an analytical to semi prep tool
UHPLC are more for analytical range.
the MPLC is more a prep range, use for purification, clean up

what do you need to do with your product after chromatography?

[zahra1634]

Thanks very much for your good information. Actually I am increasing my knowledge of those methods to fine the best choice. the goal is mostly analytical measurement to find the conjugation yield. I think SEC or RP-HPLC might work but I am not sure about the solvents yet. I would appreciate it if you could suggest any way that would be compatible with protein and DNA at the same time.

Zahra

[ScottHorn]

How big is your protein, and how big is your fluorescent tag?
As for RP-HPLC, I don't think many proteins make it through in their native state. If your protein is much larger than any free label that might be left over, SEC would probably work well for both analysis and prep. There are plenty of (relatively) cheap polymer materials that you can pack by yourself and still get good separation at prep scale. There would be no need to use any special solvents, unless your protein requires them for solubility.

Scott Horn

[unmgvar]

knowing the MWs of all 3 parts will help.
also the characteristics of each.
do you intend to modify them?
for example playing around the protein type or the oligo-chain length or type? if yes then this would complicate your situation and actually bring the SEC as the preferable choice of chromatography to simplify things,
but the info on the questions will help you decide

[zahra1634]

How big is your protein, and how big is your fluorescent tag?
As for RP-HPLC, I don't think many proteins make it through in their native state. If your protein is much larger than any free label that might be left over, SEC would probably work well for both analysis and prep. There are plenty of (relatively) cheap polymer materials that you can pack by yourself and still get good separation at prep scale. There would be no need to use any special solvents, unless your protein requires them for solubility.

Scott Horn

Thanksss Scott, my protein is about 30kDa much larger than the tag and DNA so I think SEC would be the choice.

[zahra1634]

knowing the MWs of all 3 parts will help.
also the characteristics of each.
do you intend to modify them?
for example playing around the protein type or the oligo-chain length or type? if yes then this would complicate your situation and actually bring the SEC as the preferable choice of chromatography to simplify things,
but the info on the questions will help you decide

Thanksss for your suggestion . that would be very helpful as the length of the oligo might be modified.

[zahra1634]

About SEC ,I was wondering if you know which one of gel based column or silica based column has better efficiency to separate for example two sample with just several kilodalton difference. Thank you for your help in advance.

Zahra

[Andy Alpert]

Oligonucleotides are well-retained in anion-exchange chromatography. If the protein has an isoelectric point less than 8, then you should be able to separate the unreacted protein from the protein conjugate easily by anion-exchange chromatography. In fact, the odds are good that you could accomplish that with a solid-phase extraction (SPE) cartridge of an anion-exchange material that's suitable for proteins. That would cost only about US$ 2.50 each. Now, if you want to vary the length of the oligonucleotide chain and then separate different conjugates from each other based on the length of the chain, then you'll need a high-resolution column.
I agree that SEC is unlikely to accomplish your separation. Typically, baseline separation in SEC requires the two analytes to differ by at least 2x in molecular weight.

[zahra1634]

Thanks Andy it was very very useful tips. I was wondering if you might know whether anion exchange chromatography conditions has any reverse impact on the native structure of the protein.

Zahra

[Andy Alpert]

No. Both ion-exchange chromatography and hydrophobic interaction chromatography are modes that preserve protein structure and activity, assuming that the ion-exchange material was designed for use with proteins (with a nicely polar coating that hides the underlying silica, pore diameter of about 1000 Å, etc.). The exception is proteins that consist of subunits that are held together by electrostatic attraction rather than hydrophobic interaction. In that case, the high salt concentrations of hydrophobic interaction chromatography will cause the subunits to dissociate. None of this is a concern in your particular case.

[zahra1634]

Thanks again Andy. It was so helpful as before. I was wondering if there is any way to develop a separation method to integrate both SEC and Anion exchange chromatography in my case to separate different DNa length conjugates . I would be really really appreciate it if you could let me know about that.

Zahra

[Andy Alpert]

Zahra -

Oligonucleotides as long as 40 bases and differing by just 1 base can be separated to baseline using anion-exchange chromatography. Oligos as long as ~ 80 bases can be separated by more than 50% this way as well. Really large oligos could be separated from each other via SEC provided they differ in size by at least a factor of 2. I presume your concern is for single-stranded DNA and not double-stranded.

[Ting Wu]

Hi Zahra, SAX would work. SEC might work depends on the size difference. Would you please suggest the size of the tag and DNA, besides the 30 KDa protein?

Ting

[zahra1634]

Hi Zahra, SAX would work. SEC might work depends on the size difference. Would you please suggest the size of the tag and DNA, besides the 30 KDa protein?

Ting

Thanks Ting. I am looking at SAX columns for their characteristics. The DNA size with tag is between 4-5 nm and protein is a monomer with 3X4 nm size.

Zahra