Wednesday 23 April 2014

Getting to Know your Sample



Proteins are a bit like people. People have common structural themes, but there are still differences. Luckily, proteins differ between each other more than people. Purification requires picking out ONE out of many.

Any protein based sample is a complex mixture, containing both the things you want (your protein) and a bunch of things you don't want to have. Before you start to plan the purification protocol it is always a good idea to collect as much information as you can about your sample. Consider characteristics such as


  • Molecular weight
  • Isoelectric point
  • Solubility
  • Stability
  • Known Functions


Knowing these characteristics for your protein and critical impurities (e.g. proteases, proteins or other biomolecules that may bind to your protein of interest, isoforms etc.) will help you to plan an efficient purification protocol, because you can use them for separating proteins from each other..



Proteins differ in the number of charged groups on their surface. They may have hydrophobic parts, affinity tag you have added or some biospecificity for other molecules. They can also vary dramatically in size. All of these properties and insights should be used in the design of the purification protocol.

Using a combination of properties makes purification efficient. This is a cornerstone of the CIPP purification strategy; (CIPP means Capture, Intermediate Purification and Polishing). This is always a good approach when you need to set up a purification protocol for your protein since each chromatography technology has its own limitations (we'll discuss in more detail in a coming post).

DHFR Example

In our DHFR project we hope to show you the thinking behind our choices  as well as sharing the consequences.
So, before we start to plan the protocol for the purification, we need to collect some information about DHFR.

So as suggested we started to plan the purification protocol by collecting some information about DHFR.
The information we found
- it is a single chain enzyme involved in the process for synthesis of nucleic acids (DNA)
- it contains 186 amino acids and only one Cysteine, so there are no disulfide bridges in the structure. 
- the polypeptide folding contains 8 beta sheets connected via 4 alpha helices
- the active site is situated in the N-terminal half of the sequence
- the molecular weight was 21.5 kDa and isoelectric point (pI) pI: 6.9

The pI is important for choosing which ion exchange chromatography technique and which conditions that should be used in your protocol. The size of the protein is important to know to choose a size exclusion chromatography medium with a correct separation range. 

To find the above information about the structure of DHFR we use the data base UniProt/Swiss-Prot as a starting point (http://www.uniprot.org/) we like this resource a lot because it is a hub for much of the information that is known about most proteins. 

Next the interesting part of the analysis (and choosing which methods to use) which we'll discuss in an upcoming post.

Remember the more you know the easier it is to set up your purification protocol and by getting to know your protein and the differences in its structure the better your purification. There are many databases that are excellent shortcuts to gaining valuable information on your protein and if you have some recommendations of your own please let us know via the comments section. 

For more on how to simplify planning & execution of protein purification download our free handbook