Hi to all,
I have prepared a protein complex made from two monomers keep together by a metal, I'd like to analyse it by reverse phase HPLC using a gradient from 2% of ACN, to 100%.
i have some evidences that this complex could be separated by means of size exclusion, can be electrophoresed,
I'm wandering if it can be possible that this complex breaks during the elution.
In general, a protein complex could come apart during a chromatographic run?
Thanks
Acetonitrile, like organic solvents in general, is a protein denaturant, so if your complex doesn't elute at low ACN it could become denatured. If this happens, it could clog your column.
ok, but does the interaction between the protein and the hydrophobic surface of the coloumn affect the comple stability?
Thanks!
Acetonitrile, like organic solvents in general, is a protein denaturant, so if your complex doesn't elute at low ACN it could become denatured. If this happens, it could clog your column.
Well, it depends that what kind of interaction is binding your complex. If you have covalent interaction then there is no problem to your complex. However, if you have hydrophobic interactions then I think it may have problem in RP-HPLC.
In fact, in RP-HPLC the proteins are attached to the column by hydrophobic interactions. The protein with more hydrophobic patches is attached to the column with more strength. The buffer is used to detach (by breaking hydrophobic interactions) the molecules from the column. More the strength of molecule attachment more buffer is needed to detach (this strength defines the elution times of different molecules).
As buffer is used as a source to break the hydrophobic interactions of protein molecules with column so I think it can affect the internal hydrophobic interactions of protein molecules also (not very sure about that).
So please be sure that what kind of interactions do you have in your complex.
Hope this will help
Proteins tend to denature on reversed phase resins, so there is a pretty good chance your complex will not stay together under these conditions.
The principle of RP-HPLC is that molecules (proteins) interact with the solid phase of the column through hydrophobic interactions. The more hydrophobic the molecule, the more the molecule (protein) is retained by the column. Typically, RP-HPLC methods will use an aqueous mobile phase (MPA) and an organic mobile phase (MPB). In the case described above, you are using acetonitrile (ACN) for MPB. What are you using for MPA? Is it a buffer or is it water with an acid modifier? An acid modifier is usually used to improve the peak shape of the analyte. The acid modifier could potentially influence the stability of the protein complex and result in denaturing the protein in addition to the ACN.
You could analyze the primary structure (if you have access) of the monomeric protein using the Protparam tool in Expasy (http://web.expasy.org/protparam/). This is an awesome tool "which allows the computation of various physical and chemical parameters for a given protein stored in Swiss-Prot or TrEMBL or for a user entered protein sequence. The computed parameters include the molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (GRAVY)". While the Protparam tool yields an estimate of the hydrophobicity of the protein based on the primary structure at least you can use it as a starting point for your RP-HPLC method development.
Not knowing the complexity or the time involved in your protein purification method, if it is laborious and time consumming and you want to ensure that you isolate native active protein I would recommend using SEC as opposed to RP-HPLC.
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