You can plug 2 superdex columns in tandem, or even three or four, this is no problem to increase column length, but I doubt this will be enough to separate such complexes (500 vs 525 is 5% MW difference, i've never heard of such resolution of SEC).

I think, all the techniques of separation based on the (apparent) molecular mass / hydrodynamic radius or other physical caracteristic of your complexes will fail.

If you can get your protein C highly charged compared to the others (choosing appropriate pH), you could try to IEX, but this is also unlikely to work and could lead to complex dissociation.

How do you get your complex ? Is it in vitro reconstituted ? If you could manage to get the protein C in a limitant amount, you could increase the homogeneity of the sample shifting to the lower content of C in your complexes.

Good luck !

Why dont you try different types of columns,, like ion exchange?

you can try antibody-affinity column if you can get antibodies for the proteins you wish to separate. This would make the process simpler and also effective. But cost could be a factor.

You should first try Ion Exchange or HIC, if you dont want to do that as it would involve process devleopment, then try a Sephacryl S-300 or S-400 sizing column. Sephacryl resin is less expensive than superdex and is more suited for the size range you are in. Start with a 60 cm length, longer may allow for better separation but try the 60 cm first...Good Luck!

I think you have to use at least S-400. A S-300 is not 'good' enough to seperate them. Yes I agree the longer the better. And start with loading less samples to see if it is possible to seperate them. Good Luck!

You can plug 2 superdex columns in tandem, or even three or four, this is no problem to increase column length, but I doubt this will be enough to separate such complexes (500 vs 525 is 5% MW difference, i've never heard of such resolution of SEC).

I think, all the techniques of separation based on the (apparent) molecular mass / hydrodynamic radius or other physical caracteristic of your complexes will fail.

If you can get your protein C highly charged compared to the others (choosing appropriate pH), you could try to IEX, but this is also unlikely to work and could lead to complex dissociation.

How do you get your complex ? Is it in vitro reconstituted ? If you could manage to get the protein C in a limitant amount, you could increase the homogeneity of the sample shifting to the lower content of C in your complexes.

Good luck !

It is very difficult to separate 500 and 525 kDa proteins. I suppose you can use ToyoPearl TSK-gel HW-55 SUPERFINE. This gel has a wide fractionation range 10 - 900 kDa . You can connect two or three columns and obtain 180 - 270 cm mega column. Please see: http://www.separations.asia.tosohbioscience.com/Products/HPLCColumns/SizeExclusion/ProteinsBiopolymers/, and http://www.separations.asia.tosohbioscience.com/NR/rdonlyres/57517595-D5CC-460D-B340-E2C4ED11710F/0/1_01E_TOYO_HW_Man.pdf

All the best!

If I were you I would use IEF (isoelectric focusing) to purify your proteins. Some proteins with similar MW can shows different pIs and that is enough to get a good separation! Just let me know if you need additional information !

Cannot be done, period, unless there were an extreme conformational difference. Rule of thumb: SEC can resolve globular proteins differing by twofold in MW. At 1.5 fold they will overlap into a single broad peak and no column length or expensive medium will improve it. Ackers treated this subject thoroughly in his 1970 Adv Prot Chem article.

I doubt myself that it is possible but 'someone' has to try anyways....so I go with the suggestions above. (Yes, it is in-vitro reconstructed and it has already been on Ion exchange chromatography)

thank you all.

I think that this separation is praticaly impossible bySEC. I suggest you try other methodology such as ion exchange chromatography.

Size exclusion will not help you, the difference is to low. You can only do it by pulldowns and then check on the SDS gel intensity of your low molecular weight protein

As Levy and others suggested, I would test the stability of the complex at high ionic strength first. If stable, ion exchange or hydrophobic interaction chromatography. The difficulty now is how to test complex formation without actually separating them? The size distribution from DLS may be one answer but I am not sure if it would be sensitive enough to pick up the difference.

Another way to do it may be to create separate affinity tags for A, B and C. You may be able to separate different numbers of each subunit in the complex under highly eluting conditions.

Purifying this complex would be an interesting biotechnology paper by itself.

If you do not want try IEF-based separation, I recommend you to use electrophoresis-based method such as Prep cell Unit from Bio-Rad. If you chose this method for the purification of your protein then I suggest you to make a “gradient gel”. In that way, the electrophoretic gel works like an exclusion column, demanding less time and getting mgs of your proteins. In both cases, you can to get native proteins even if they come as a complex.

http://www.bio-rad.com/LifeScience/pdf/Bulletin_1964.pdf

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1702950E.pdf

Good luck !

Thank you all again, you are too kind and full of useful information.

First thing I will probably try is borrowing a Mono P column from a colleague. I stumbled on chromatofocusing when I was reading on liquid phase IEF (Thanks again for the hint).

Hi Stefan,

I agree with the colleagues above - use IEF (e.g. ZOOM IEF Fractionator), or the Prep cell, or maybe native electrophoresis ... Do you have a possibility to produce antibodies against the particular proteins? Immunoprecipitation could also help.

Hi Stefan,

As said by others SEC may not be helpful in achieving the resolution you are looking for. If both the complexes have differences in Hydrophobicity, resolution can be attained after ammonium sulphate fractionation. First check with number of hydrophobic aminoacids and distribution of the same. Try with different cutoffs of ammonium sulphate fractionation and find out whether the desired protein complex is coming in pellet or supernatant after fractionation and go ahead accordingly.

Goodluck

Hi Stefan,

If both proteins will have different pI then you could able to separate them on the basis of their pI in IEF and later on you can check the mass by using mas spectrometry

I agree with Rajdeep and Whyte. A size/mass separation will not work. But the complexes could have radically different charges, and might be separable by charge. We like mixed bed ion exchangers for high resolution separations, but other columns are available. So, the answer is 'no' and 'maybe'. Some pragmatic experiments are called for.

he wants to separate 500 from 525, unless these proteins form s-s bonds, using ion exchange or IEF is not going to work. he may be able to do some native page using agarose.

Separating 500 and 525KDa by mass requires a native protein separation on the basis of mass/hydrodynamic properties with a resolution of 25kDa in 500kDa - or about 2.5%. Impossible! However, if the complexes have different charges, then a clear separation might be completely feasible. As I've suggested, some pragmatism is called for here. Native agarose would be charge based separation, although I would prefer ion exchange as this moves the complexes into absorption/desorption and provides peak sharpening if a gradient is employed. As an extreme chromatofocussing might be best?

I think you have very nice suggestions and expert inputs above. For joking, maybe you need a skyscraper column for that! I don't know what is the number of theoretical plate you will need to separate them in SEC provided ideal conditions.

I just want to ask even if somehow you succeed to separate the two complexes, how can you demonstrate that it is not an artifact of the preparation as they bear the same units with only differences in their stoichiometry? I mean whether or not these two complexes really exist in the cell

If the two complexes migrate through a native gel as separate, distinct bands, then PrepCell (Bio-Rad) may be another option.  I have not looked carefully at other suggestions, but I would first try ion exchange chromatography, using the one that only binds to component C.  You may be able to separate the two complexes by a gradual salt gradient (complexes containing more numbers of component C would elute later in the gradient).  Gook luck with your experiment.