Hi Claire,

First question: from which source do you isolate your crude protein lysate/complex (bacterial, yeast, plant, mammalian...)? And I agree Martin, S200 gel filtration is probably not the best choice but you could be lucky.

Answering the concentration problem: I think, the best concentration is the highest you can reach. in gel filtration the sample volume is more critical than the concentration, however, you should check complex stability @ high conc beforehand.

Consider as first purification step a fractional ammonium sulfate precipitation(group fractionation), because the method is non-denaturating ending up with low sample volume (and high conc) and you get rid of some impurities - that will probably enhance complex stability.

Hi Claire,

it is hard to know beforehand whether your complex survives sonication. This depends very much on its stability, as well as things like buffer composition etc.

However, your complex will run in the void volume of the column, together with possibly many other complexes, so an S-200 SEC to purify out of lysates is probably not an optimal choice.

Hi Claire,

First question: from which source do you isolate your crude protein lysate/complex (bacterial, yeast, plant, mammalian...)? And I agree Martin, S200 gel filtration is probably not the best choice but you could be lucky.

Answering the concentration problem: I think, the best concentration is the highest you can reach. in gel filtration the sample volume is more critical than the concentration, however, you should check complex stability @ high conc beforehand.

Consider as first purification step a fractional ammonium sulfate precipitation(group fractionation), because the method is non-denaturating ending up with low sample volume (and high conc) and you get rid of some impurities - that will probably enhance complex stability.

Hi Martin and Lars,

Thanks very much for your speedy responses - very grateful. We are eventually hoping to look for a caspase activation complex isolated from brain injured mouse cortex/hippocampus, but thought we'd start off with stressing some primary cortical neurons and making lysates from those.

I'm confessing to a slight laziness in that the choice of the S200 column was entirely down to the fact that there is one already in the dept, attached to an AKTA! I was, as you correctly surmised, hoping to get lucky with the elution fraction. I was also considering a concentration step on a spin column with a 100kD MWCO beforehand to reduce the complexity of the buffer to something like PBS and to eliminate some of the smaller MW proteins. This would also mean I could load at a high conc, but didn't want to overface the (dept's) column. Would amm sulph be a better option than a spin column, Lars?

A final point in this rather epic response - I am also considering running a native gel prior to the column so that I can see that I have retained some high molecular weight complexes containing caspase. Do you think this is worth it or will work for up to 600kD?

In this case, I suspect the complex concentration will quite low and ammonium sulphate will not be a very good option (you need reasonably high protein conc). In this case I would go for spin columns as well, or a combination of both methods, if the you encounters protein aggregation during concentration (first spin then ammonium sulph).

I thought also about a native gel, blotting and subunit detection, but I'm not sure about running properties of the complex. It might stick in the well on the top of the gel and not move into the gel... give it a try, when you have enough material.

I agree with Lars' idea of gel elctrophoresis. If you have the option to run native gels, or even native 2D gels, this would be a nice way of assessing complex stability.

Thanks both. Yes we have the native gel stuff on order so will give that a go and if it works, at least we will know whether our complex is stable through sonication (I'm assuming if its not, then I will see smaller mass caspase signals in the sonicated compared with the non).

If you had a free choice, what column do you think would be the best for this type of experiment?

I think you might only be able to enrich for your complex if it does not carry any form of affinity tag.

This leaves only physicochemical properties (size, pI, hydrophobicity) for purification. If your complex is stable in slightly higher salt concentrations, ion exchange chromatography can be an option. GE offers a HiTrap IEX Selection Kit, where you have a number of column media options to choose from. Once you have some enrichment, you can still move to size exclusion.

Link for the column set: http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/de/GELifeSciences-de/products/AlternativeProductStructure_17470/17600233

Thanks Martin and Carsten, will try adding glycerol to the buffer then and aim for a 5mg/ml conc. The column set also looks quite interesting and not as pricey as I though it might be. If I don't get lucky with the superdex, I will definitely try the set. Will let you all know!

Thanks again for all your help,

Claire

We have had a lot of success in the past few years using the blue native gel system. It will depend upon the abundance of your complex and whether you know components to use in immunoblots to find it. But if your goal is to identify components in large complex, the blue native gel has far better resolving power than other native gel systems or gel filtration and can be used directly for LC-MS/MS id of protein components

Considering that you don't seem to know much about the properties of the complex, I would go with the most simple approach (lysis, load and run gel filtration colum, analyze fractions), to avoid problems by introducing to many steps. If that is not working out one can, one by one, introduce additional steps. Lysis: if you want to avoid high detergent conc., one can also use a small 27G needle and pass the cell suspension through it with a small 1ml syringe ("no dead volume" type) 20-30 times; this may be less harsh than sonication. Regarding concentration: the gel filtration (Sup 6 would also be my choice; but you can try Superdex 200 first, if that's what you have attached to your system) will dilute your sample approx. 10 fold (depending on the fractionation profile of course). However, if you can detect your complex in the input extract by westernblot against one/some of the subunits, you should be fine. You can always concentrate the fractions that you are collecting 10 fold, by TCA precipitation for example, and run the westernblot to detect your complex.

Btw. a very mild alternative to gel filtration for very large complexes would be sucrose gradient centrifugation. It has less resolution, but it is much less prone to problems like aggregation etc.

Thanks Richard and Jens. Richard, by a curious co-incidence, it's the blue gel kit we went for when purchasing a native gel in order to see if sonication was going to disrupt the complex. Have you digested and mass spec'd straight from the native gel then?

Lars, we have carried out a test lysis in detergent-free conditions using a 27G needle and found that the yields were much lower than if we'd sonicated or used detergent. We also tried freeze thawing a couple of times but the yield was still poor in contrast with the sonicated sample, hence wanting to use this in our protocol. We were planning on blotting for stupidly high molecular weight caspase complexes using the native gel, but from RIchard's comment (if I have it correctly) it sounds as if we might be able to use the gel directly to isolate and identify the complex.

I recently tried to help a nearby lab purify a complex with a similar size range. Gel filtration ended up being a little tricky and we never obtained a complex. They ended up going the route of sucrose gradients like Jens suggested. The sonication question is an empirical mater, however, 3 x 10 sec isn't a very rough procedure. Maybe you could look into coupling cross-linking with the above suggestions?