Structural Biochemistry/Proteins/Purification/The Miniaturization of PFK-1 Purifications
We are moving from Standing Purification to Micropurification, which is 100th of the volume of standard, and there are many reasons that scientists are shifting toward the micropurification. A major reason is biochemists are seeking to ultimately work on cancer cells. However, They are expecting that Cancer cells that are provided will be small. Therefore, it has been crucial to modify the existing purification (standing purification) to Micropurification, which is 100th in volume.
Another reason is that biochemists are going to find a suitable capacity of DEAE Sephacel beets to the volume of proteins, meaning they are going to saturate the beets with proteins to find the maximum capacity.
Standard purification of protein is considered kind old school of biochemistry techniques in which proteins are separated based on their properties such size charge solubility functional groups and etc. In sanding purification, biochemistry usually start with source of protein and by using different combined techniques of separations, they were able to discriminate between proteins based on their properties such centrifugation and electrophoresis. In Micropurification, It is similar to standard purification but biochemists modified the scale of the stands purification. For example, in DEAE instead of using 50-60 ml of DEAE-Sephacel Ion-exchange column, biochemists could use .1 ml. generally speaking most of the scale of the techniques employed in Micropurification is miniaturized.
Methods and Materials
Researchers used micropurification technique in variety of proteins however; we will focus more in the micropurification of Phosphofructokinase-1 which is a protein that regulates the glycolysis process. In the first step, usually is to obtain the protein from a source, in this case the source was rabbit muscle tissue. Then, the muscle tissue is being blended and homogenized. However, in order to keep the proteins from decomposition, biochemists usually use chemicals that acts as a protease inhibitor to protect the proteins from being decomposed upon homogenization. The homogenate is then balance.
The centrifugation step follows the homogenate step. In this step, biochemists usually separate the supernatant and the precipitate by density. PFK-1 is a soluble protein so we expect it to be in the supernatant. Therefore, we can just discard the supernatant. At this point, this we have many stuff in the supernatant fluid besides our protein.
Just like standard purification, the next step will be what is called the heat step. Heat de-naturation is performed when the solid material is re-suspended. The purpose of this step is to denature all the proteins that are in the solution and not our protein of interest. This could be done by using a solution that contains the substrates MgSO4 and ATP that acts to stabilize rPFK-1 protecting it from denaturation. Then, we separate the supernatant and the bullets and we expect our protein to be in the supernatant.
Affinity chromatography technique is employed to separate proteins according to their charge. After the heat-step, the 1cc micro-purification columns are set up according to standard purification procedures using 1/10 the volume of DEAE (ion-exchange) beads to separate the enzymes. Since our protein is positive charged and the beads are negative charged, our protein adheres to the beads. After that, addition of a successful portions of weak base is used to with different concentrations. Finally our protein is obtained with multiple fractions.
Finally, Lastly, if for any reason the purification could not be completed in one day, an additional step of salting-out is required to precipitate the enzyme. A G-25 Sephadex desalt column composed of beads that separate the enzyme from the NH4SO4 by size. Ideally, the first few fractions collected are expected to contain the greatest amount of PF Works Cited Russell, P.J. "Inhibition of rabbit muscle isozymes by vitamin C." the Harwood Academic Publishers imprin 15 (2000): 283-296.
K-1 activity with the least amount of NH4SO4 present. To check for the purity of the protein biochemists used SDS-PAGE. In this step we were able to identify how pure our enzyme was and its molecular weight in Doltons. Refer (Russell)Works Cited Russell, P.J. "Inhibition of rabbit muscle isozymes by vitamin C." the Harwood Academic Publishers imprin 15 (2000): 283-296.
, A. Williams, and T.A. Austin ,(2000) J. Enzyme Inhibition, Vol. 15, 283-296.
P.J. Russell, A. Williams and D. Gapuz (1997) Biochem. Biophys. Res. Commun., 233, 386-388.