how can elution buffer elute proteins by changing ph?what happens by changing ph?

Answer 1

An excellent question! OK, first I assume you're speaking of ion exchange chromatography. Ion exchange resins work by having a fixed charge on their surface (let's say a positively-charged group like a quaternary amine) which, before you add protein, is neutralized by soluble counterions (like chloride, in this case) from the liquid phase (the buffer). As you probably know, most proteins contain charged amino acids on their surfaces. Even if proteins have an equal number of positively-charged and negatively-charged amino acids on the surface, they're never exactly evenly distributed which means that there are areas on the surface of the protein which have an overall positive or negative charge to them.

Now, in this example, negatively-charged patches on the surface of the protein will be attracted to the quaternary amines on the resin (displacing the chloride counterion in the process). At this point the protein is bound to the surface of the resin (or bead, if you like).

Now lower the pH. On the surface of the proteins, the negatively-charged amino acids become protonated, losing their charge. Other groups, such as histidine, which might have been neutral, can now become protonated, picking up a positive charge. The overall result is that the protein becomes less negatively charged and so will bind more weakly to the quaternary amines, or even desorb completely. What about the quaternary amines? Well, these groups are already positively charged (as noted above) and cannot become *more* positively charged due to their structure, so nothing happens to the charge of the resin. Presto, you get protein elution!

The exact reverse happens with a negatively-charged resin: proteins bind by virtue of their *positive* charges on their surface, and as the pH is *raised*, these groups *lose* protons and become either neutral (like histidine) or negatively-charged (like aspartic acid), making the protein overall more negative and, once again, allowing it to desorb from the resin.

Answer 2

Protein A Elution Buffer

Answer 3

I assume you are talking about eluting a protein off of beads or off of a column? The column material is made of a material that will make proteins stick to it under certain conditions. Some are hydrophobic and will cause the more hydrophobic proteins to stick to it; some are charged and caused highly charged proteins to stick to it. In your case, it is most likely binding amino acids that change binding activity as the pH changes.

In eluting the protein, you are adding various different concentrations of buffer which will eventually make the protein fall off the column. The buffer usually takes the place of the column material (organic solvent for hydrophobic, ionic solution for charged proteins) and fills the site that was binding to the column before with buffer. This is why the protein comes unstuck from the column.

Answer 4

Proteins have both positive and negative charges. At different pH environments, their net charge will be different. I assume you are talking about ion exchange column chromatography. A pH gradient will elute different proteins according to their net charges.

Answer 5

certain protiens work or live in certain environments, so if u change the environment, by say changing the ph or temperature, they dont work as well or even denature.