Lab 4 Flashcards
what is the difference between analytical and preparative experiments?
Analytical is all about examining the molecules of interest but preparative is about purifying the molecules of interest
What were the order of the steps for purification?
B-gal purified from a cell-free lysate by ammonium salt precipitation first, then through size exclusion chromatography and ion exchange chromatography
How was purification analyzed?
SDS-PAGE, Bradford Assay (protein concentration determination) and enzymatic activity assay
What does ammonium sulfate precipitation separate based on? How does it work?
Separates based on differential solubility. You create a pellet of protein and the more ammonium sulfate salt ions, the more those compete with water and the greater the precipitate. Different proteins precipitate at different concentrations of salt.
Where can we find the B-gal after the ammonium sulfate precipitation? Why is this step done first and what must be done afterwards
We expect to find it in the pellet (not in the supernatant) and it is done first because it is good for initially concentrating proteins and purifies. Since the sample then contains a lot of salt it needs to be purified using dialysis or SEC.
How does dialysis work and what step WOULD it be done after (not done during this lab)
Dialysis is that the solubilized proteins are added to a cellulose membrane and submerged in buffer. Small molecules like salt diffuse out to equilibrium while proteins stay in. Changing out the buffer will lead to only proteins in the cellulose membrane.
What is SEC used for in this lab? How does it work?
Separates by size and shape (remove salts and separate by size here). It has beads in a column and if the proteins/salts are small they will fit into the beads and stay there (in the stationary phase) if too big will be excluded (in the mobile phase).
When will B-gal be removed from the column? Why?
The B-gal will be removed from the column early on because the beads exclude molecules greater than 150,000 Da and because B-gal is 465,000 Da then it will be excluded. The ammonium sulfate will stay in the beads.
How does the Ion-Exchange Chromatography work in the lab? What does it separate by?
It separates by charge. In the lab we have a anion exchange column with covalently bound positive charges. A pH or salt gradient can be used to elude the proteins but here we use a salt gradient. The pH of the buffer is held constant (at 7.5) and use increasing salt concentrations (0.2 and 0.4 respectively). The salt competes with the proteins for space.
What is specific activity?
The micro-mol of substrate cleaved per minute per mg of protein. An increase in this value shows successful purification
What tests are used for analysis of purified B-gal?
B-gal activity enzyme (using ONPG), SDS-PAGE and Bradford
What is needed in the B-gal activity enzyme for rate of ONP being proportional to amount of enzyme present?
Substrate is non-limiting, have necessary cofactors (like Mg2), and the temp pH and ionic strength are not denaturing
What is the Bradford used to determine in this lab?
Total protein (in lab 3 used to calculate the concentration of the unknown)
How does the Bradford assay work?
The Coomassie Brilliant Blue will bind to tryptophan, tyrosine, arginine, phenylalanine, and histidine (ttaph), with a shift in absorbance from 465nm to 595nm due to stabilization of the anionic form of the dye.
For SDS-PAGE the samples are treated with sample buffer, what is in the sample buffer and what does each component do?
Glycerol: increases density of the sample
B-mercatoethanol: reduces intra- and inter-chain disulfide bonds
SDS: anionic detergent that unfolds polypeptide chains and coats them with negative charges
Bromophenol blue: tracking dye that migrates faster than the proteins in the sample
