Buffer for Protein Purification

This Wednesday (April 17, 2013) I prepared for some buffers for protein purification with Namita. We made four 200 ml buffers:

1. Lysis buffer - used to lyse the bacteria in order to collect the proteins. The solution would contain all proteins the bacteria produce. 
2. Wash buffer (20mM) - used to wash out some undesired proteins
3. Wash buffer (150mM) - further wash out the undesired proteins by breaking bonds between undesired proteins and resins
4. 300mM - 300mM imidazole solution can break the bonds between the targeted proteins and Ni. After washing away other proteins, the column by this time contains mainly the targeted protein bonded with nickel. 

Materials

• H2O
• NaH2PO4
• NaCl
• Imidazole 

The buffers only differ in their percentage of imidazole. Imidazole is used to separate bonds between nickel and proteins. The targeted protein is his-tagged, which have a high affinity to nickel when running through the column. However, some random proteins can also loosely bind to Ni too. The higher the concentration of imidazole, the stronger bond it can break. Here 300mM is the concentration to break the bond between our proteins and Ni.

To determine what concentration breaks the bond between targeted protein and Ni, one runs a gel to determine the size of the target protein and at what [mM] does the solution contains the most targeted proteins.

Example of a protein gel.
Retrieved from http://www.sciencedirect.com/science/article/pii/S0168165610001926

For example, as shown above, if lane 1 is 10mM and increases by 10mM each lane. At 80mM (lane 8), a clear dark band is shown, meaning that 80mM of imidazole breaks the bond between OmpA70 and Ni the best. Othe lower concentrations are used to washed off some contamination shown by the blurring bands in lane 1-7.

After we added the appropriate amount of substances into four flasks according to the calculation, we need to make the pH into 8. We do this by adding NaOH to the solution and using a pH meter to measure the pH.

pH meter (right) and magnetic stirrer (left)

It took us quite a long time though, especially the two with higher concentration of imidazole bacause imidazole is slightly acidic. Luckily, we have the magnetic stirrer, which i thought was a very brillant invention, to speed up the mixing rate.

Magnetic stirrer

When all the buffers finally reach pH8, we decided to call that for a day. Making buffers, as Namita admitted, can be very boring, yet it is very demanding because everything should be very concise. Later, Namita would use those buffer to establish a  imidazole gradient to collect and purify the proteins from E.coli. For the details please look here.

Where does this fit on our map?

P.S. I will post the blog from last week asap!