Can I use chaotropes or detergents in my buffers when doing ion exchange chromatography?

Yes. But be sure to use non-ionic detergents and chaotropes. Urea works well for ion exchange. Beware of guanidine salts as they are salts and will not allow your protein to bind.

Help, my protein is not eluting off of my ion exchange resin!!

That's not really a question, but there are a few things you could try: Proteins can be much more soluble at high pH, so try pH 10-11 and elute with salt. If it does not like high pH try low pH - discharge the resin at pH=3. You may also be able to rescue it if it's refoldable: Just rinse the column with 6M guanidine hydrochloride. Your protein is probably stuck non-specifically to the support to which the functional groups are attached. Try a different support. Rumor has it that heat and mild detergents may also get your protein off.

Which direction will my protein travel when doing a blot after SDS-PAGE?

Your protein will travel away from the negative pole, towards the cathode, the positive lead (in most cases the "red" wire or side). This is due to the fact that your protein after SDS-PAGE is coated by the negatively charged SDS molecules and will migrate to the positive pole. Be sure to give your protein enough time to exit the gel and enter your blotting matrix.

Where is my protein? I can't find it after reversed phase!

You may have used a matrix which is too hydrophobic to allow your protein to elute in a standard acetonitrile gradient. If you have more sample try a matrix with less carbons (i.e. C4 instead of C18) or with more polar atoms (CN versus C4). If you REALLY need the sample you loaded, it may elute after washing with isopropanol, or formic acid, acetone, chloroform, ethanol...

Why are my large proteins eluting before my small ones on a size exclusion column - shouldn't the smaller ones just slide right through?

SEC relies on the volume a molecule can travel through. The more volume it has to travel through, the longer it will take to elute. SEC resins have tunnels of various sizes, the range of which are given by the manufacturer for theoretical, globular proteins. Small proteins can access a large amount of these tunnels and take a longer time to come out, while larger ones in extreme cases do not even enter any of the beads and snake their way through the beads. Thus, they see little of the total volume and take less time to elute from your column.

Will my protein always have a Met on the end when expressing it in recombinant systems?

No. In many cases you will not have a Met left after processing. Proteins expressed in E. coli have the Met removed most of the time when a small residue follows the N-terminal Met. In some eukaryotes you will never see you Met. These removals are catalyzed by N-terminal methionyl peptidases which are present in most cells. Don't fret without the Met though: It is highly likely your native protein doesn't have it either!

How can I improve the resolution of my ion exchange chromatography separation?

IEC affinities rely heavily on pH: Try to change the pH at which you elute or instead of using a salt gradient do a pH - step gradient. Mix buffers with different pHs decreasing the pH by 0.2 to 0.5 units for each buffer. Elute going from high to low pH. Make sure you load slow enough for the protein to diffuse and contact the resin, but elute at a faster rate with at least 20 to 40 column volumes of buffer in you gradient. Also, modify your gradient! Flatten it out where our protein elutes. Peaks will broaden, but the resolution will increase.

Help my protein is not binding to my ion exchange resin!

Does your peptide have charges? Increase the pH if using anion exchangers (Q, DEAE); Decrease it if using cation exchangers (SP, CM). Your protein not binding my be a good thing if all the other proteins are you have a simple, effective purification.

What is a FAQ?

A FAQ is a list of Frequently Asked Questions. This FAQ will keep a record of the most commonly asked questions about protein chemistry and will post the answers received as well.