Additive

[danko]

I’m having trouble with a huge protein molecule that sticks to the stationary phase.
Even the most polar (cyano) column I could find holds on the compound and no amount of acetonitrile is big enough to elute it. I’m aware of other sub- techniques such as GPC and IE, but I’d like to try reverse phase because of its resolving power.
Anyone has an idea of what could be added to the mobile phase in order to elute the compound?
Salts are not an option, because they precipitate the compound.

Best Regards

Danko

[tom jupille]

First off, is your protein soluble in acetonitrile? HIC or IEX might be much better bets.

[james little]

Someone at ASMS in one of the workshops was really showing nice data on monolith columns. Especially with respect to minimizing carry over..

[danko]

Hi Tom Jupille,
And thank you for the response. It’s not soluble in 100 % acetonitrile, but soluble in 80 %.
And that is the proportion I tried to elute with at the end of the gradient. Unfortunately without success.
As I mentioned, salts precipitate the compound, so HIC and IEX are not plausible alternatives.

Best Regards

Danko

[danko]

Hi Sailor,
Thanks. It sounds like a very good idea. I’ve never tried monolithic columns, so maybe it’s about time to investigate this option.

Best Regards

Danko

[HW Mueller]

There will always be some salts (chaotropic), or some low concentrations of about any salt, which will increase almost all protein solubilities, or chaotropic substances like urea...
Your problem could be that 80% ACN doesn´t de-adsorb the protein from your stat phase, soluble or not.

[tom jupille]

Expanding on Hans's comment a bit: precipitation in salt solution is exactly what HIC is based on. You start at a relatively high concentration to make the protein stick and then decrease it until it re-dissolves.

[danko]

Hi Tom,
I know how HIC works, but you se, the substance stiks to the stat. phase without any salt, so why should I start with a high concentration of salt?
But anyway I appreciate all suggestions.
Urea might be the solution, but I think it is a tricky substance to introduce in the HPLC system.
By the way. This forum is a very nice place to be. I'm new here and I like it a lot

Best regards

Danko

[tom jupille]

I know how HIC works, but you se, the substance stiks to the stat. phase without any salt, so why should I start with a high concentration of salt?

Sorry, I didn't elaborate enough:

• 1. Typically, HIC packings are purpose-made; they are much less hydrophobic compared to RP packings.
  2. HIC is usually a non-denaturing technique, whereas RP is almost always denaturing. This means that your protein is likely to be more hydrophobic under RP conditions (as a generalization, "native" proteins tend to have hydrophilic groups on the surface, denatured proteins tend to have hydrophobic groups on the surface).

Bottom line: the fact that your protein sticks tightly to your RP packing is not a guarantee that it won't elute nicely in HIC.

[HW Mueller]

The only thing funny about urea is that if you are sloppy you will have white crytals all over. There are also detergents (have only experience in cleaning columns, I avoid them as they are difficult to get out of columns) and chaotropic salts. Also, you probably drastically need to reduce the ACN or use alcohols, isopropanol works quite well with some proteins. If the suggestions made so far don´t help, look at what membrane protein researchers do.

[danko]

Hi Guys,
The propanol helped a lot. I had to add some glycin though (10 mM).
I’d like to thank you for your contribution and underline my pleasure to chat with you.

Best Regards

Danko