Hello, I'm an undergraduate. First of all, I have a few questions about FPLC which are of proof of concept, not that I'm looking to detect a particular protein. I would like to know if a High Performance Liquid Chromatography (HPLC) instrument can perform Fast Protein Liquid Chromatography if the columns are changed, or would pressure conditions not comply with such a change? I think that the metal columns of HPLC instruments may conduct heat as the mobile phase passes through the column which may initiate denaturation of heat-sensitive proteins in solution upon contact with the column. However, there are research papers which use HPLC to detect proteins, so it must be possible to work around these two problems in proteonomic analysis. So if it's possible to use HPLC instruments, why would scientists choose to use FPLC instruments? What FPLC instruments and/or instrument suppliers would you recommend? Of course, multi-wavelength detection and the ability to couple chromatography columns such as ion-exchange and affinity in series should increase the separation potential of the instruments. However, these novelties also increase the price. I know that GE Healthcare Life Sciences has several FPLC models available, for example.
To make the most of your time, if you wanted to isolate a particular protein with FPLC from a large matrix of proteins, how would you do it? If, for example, the protein was lipophilic, greater than 1,000 Daltons in size, proline rich, using manganese as a cofactor, stable within a small basic pH range, how would you isolate it?
It would occur to me that the protein could be isolated by elution within its small pH range, and a polyhistidine tag could be cloned onto the protein so that it could bond to a zinc stationary phase by affinity chromatography. However, how would you exploit its lipophilic nature? Could there be a more creative approach than simply using a polyhistidine tag?
Thank you very much. As my question is conceptual if you could provide me with any research papers or review articles to illustrate your point, I would love that!
Thank you.
the difference between FPLC to HPLC lies mainly in resolution and capacity. HPLC and UHPLC are used extensively for protein chromatography. Laboratory based HPLC normally operate up to semi-preperative level (i.e. ~5mg total protein), while FPLC is used for larger quantities - especially when purification of expressed protein from large quantities of lysates are being run.
FPLC are also less prone to blockages, especially for SEC columns, which are often self-packed.
there is no real problem running FPLC column on HPLC system as long as you make sure not to go over the pressure limit. it will just be very inefficient.
David.
The resin design for FPLC was a nice marketing plan by Pharmacia/ Amersham, now GE Healthcare. Why not make chrom. resins that have better resolution than the soft gels that existed back then, make the resolution try to approach that of analytical/preparative HPLC, have greater capacity than HPLC (as David points out), but then force investigators to purchase the necessary higher pressure instrumentation with programming/automation like the HPLC's but at a much lower cost than HPLC's which many investigators didn't have back in the day? The resin base-media became more cross- linked like today's Capto series along with significantly smaller bead size of narrow distribution that do make a difference (1/square root particle size diameter), very large pore size with narrow distribution relative to silica based HPLC resins, better bead morphology, but not too much of a change in alpha value (selectivity).
However, FPLC media can and is used on HPLC's for analytical applications. An example is tandemly linked Superdex 200 analytical SEC columns used in cases where the protein has adverse interactions with the base matrix of other resins that can't be resolved with solvent changes while maintaining good SEC behavior. Just be patient and run it at the recommended pressure. Regarding your question about alpha value for lipophilics, indeed there are FPLC resins that are similar to reverse phase in terms bonded phases and these are the HIC resins which exist in many flavors, your choice.
The hydrophobic 1kd Mn-protein that's stable in a narrow ranged alkaline pH may not require a His tag if the proteinaceous contaminants are not extensive including product related contaminants. But if you do use His and the product is for in vivo work then use Zn. Ni is poisonous. You would also need to know which end of the protein to tag. I don't think Mn will interfere knowing the orbital structure but you might want to keep Mn present in all solutions for stability. You need to know the pI of your protein and the amino acid (aa) sequence to help determine purification unit operations and formulation stability. Proline richness is partially helpful in purification strategy since the conformation may be fairly rigid and could change under low pH conditions due to isomerization. In other words the Pro's cis/trans could give conformational heterogeneity to begin with and cause some inactivity and different exposed charged residues that lead to multiple peaks on ion exchange Chrom. The aa seq is extremely helpful and an important aa is cysteine. The molecule could be a natural covalent or non-covalent multimer or a free thiol may lead to adverse aggregation/ instability. Again, the aa seq is needed and gives the info for charge, hydroph/hydrophilic, size, possible confirmation, multimerization , potential co-factor req, stability,
Hi,
As your protein (peptide) is so small and lipophilic then you can use either reverse phase HPLC columns or HIC with FPLC columns. HPLC will obviously give you much better final resolution but for initial puriifcation/enrichment you could try HIC first to remove the majority of the contaminating proteins.
IEX may also be an option to remove other proteins and peptides prior to your HIC/RP steps-either leaving your peptide in the flowthrough or enriching it on an IEX column depending on charge etc. but if it is very hydrophobic probably the former is the way to go.
You don't say if this peptide is heterologously overexpressed or endogenous in a biological sample, if it is endogenous then the tagging option is out. If it is for over-expression you could try something like the his-SUMO tag followed by cleavage which is a very efficient way to produce peptides.
The sequence of your protein is extremely important in terms of anticipating and explaining its behavior in purification, stability, formulation, phamacokinetics, drug metabolism, expression, manufacturability, etc. Are there cysteines in the sequence which may require a folding protocol or buffer development to prevent covalent oligomer/aggregation formation unless that is supposed to occur or perhaps folding is influenced by Mn as in a metalloprotein versus a metalloprotein cofactor? There may be cleavable fusion protein strategies to consider in terms of expression level and purification.
There are a lot of confusing terms developed by marketing people to try to 'guide' (trick) people into some narrow alley of their choosing. For beginners in the field, it is just a lot of confusing misinformation, unfortunately.
All of liquid chromatography is done with variations which are engineered to meet the time, capacity and resolution requirements of the application. The key elements to consider for engineering are surface area and mean analyte travel path and practical constraints of cost and size,
HPLC, for example is nothing but old fashioned column chromatography using smaller particles and decent mechanical engineering to take care of the practical details caused by using the small particles needed to give the high separation and capacity per unit time. Flow injection analysis is just a flow system like an hplc, but without the column.