What type of column is used in the present experiment? What is the name of the process? What does a column usually have in it? What does the present column have in it? What type of molecule is it?
- IMAC (Immobilized Metal Affinity Chromatography) is the name of the process.
- A column with material packed in it.
- In the present experiment, the column also has nickel Ni+2
- Nickel is a divalent cation
What will add to the column in purification? What will happen to the protein with the column? What is required for it to happen?
- Will add crude extract of the CR enzyme to the column.
- The protein with the his-tag will bind to the nickel column, while all other things will go through the column
- However, the his tag needs to be outside of the protein for binding to occur. Can change where the his tag is positioned.
What are the two important things about working with a protein? Will the protein be pure after all those steps?
- Two important things about working with protein: 1) it should always be on ice 2) should always use buffer, not water.
- Protein may not be pure even after all steps
What are the four main steps of protein purification?
Step 1: Pour the crude extract into the column
Step 2: His tagged CR protein will bind to the nickel in the column. A nickel and his tag protein complex will be created.
Step 3: Wash the column with a buffer with a low concentration of imidazole.
* Necessary for: getting rid of unwanted proteins (ones without his tags) and proteins loosely bound to the his tag.
Step 4: Elute with buffer with a high concentration of imidazole. Need to remove imidazole from protein.
What is in the elution buffer? What happens when it is in high concentration? Why is it used? What will happen to the column and protein?
Imidazole
In the high concentration the bond between the hist tag and nickel is broken
The his tag (histidine) ring structure is similar to the imidazole structure
The bond between the nickel and the histdine will be broken
What is the goal of purification of CR gene? What can we do with it?
Goals: Purity (pure protein) – quality (active pure protein) & Yield (Quantity)
What is done before purification?
Cell lysis is done before purification
What is part 1 of protein purification?
- Dissolve the appropriate amount of buffer
- Break the cell by mechanical means
- Centrifuge: Obtain Cell debris and supernatant
What are obtained in Affinity chromatography?
Obtain crude extract and supernatant
What are the two goals of protein purification? What can happen with a yield that is not good? What do you desire, what is related to? Are they proportional? Do they cross?
- Want to obtain a highly pure protein. Purity of the protein should be good and should be a large quantity (high amount of protein pure protein).
Desire high yield. Yield is very important. Is related to quantity. - Need active proteins (with structure intact, in present case tertiary structure). Is related to quality.
Can have a high yield with good purity but they can all be dead and therefore useless.
Quality and quantity are not always proportional. Can have a low quality and high quantity and vice versa. However, there is a point where both cross and align.
What is used and very important for determining the quantity and the quality of the purified protein?
Use purification table
What is the first column in the protein purification table?
Step
What is the second column in the protein purification table?
Procedure
What is the third column in the protein purification table? What are the units?
Volume (ml)
What is the fourth column in the protein purification table? What are the units?
Protein (mg/ml)
What is the fifth column in the protein purification table? What are the units? How is it calculated?
Total Protein (mg). Protein (mg/ml) x Volume (ml)
What is the sixth column in the protein purification table? What are the units?
Activity (U/ml)
What is the seventh column in the protein purification table? What are the units? How is it calculated?
Total Activity; Units (U); Volume (ml) x Activity (U/ml)
What is the eighth column in the protein purification table? What are the units? How is it calculated?
Specific Activity;U/mg; Activity(U)/Total Protein(mg)
What is the ninth column in the protein purification table? What are the units? How is it calculated?
Purification Fold; SA @ step/ SA @ step1
What is the tenth column in the protein purification table? What are the units? How is it calculated?
Yield;( TA/step/ TA step 1) * 100
In step 1, what are the purification fold and yield?
1, 100%
What does the specific activity help with? What should happen to it?
helps to understand whether purification is working (must increase to indicate it is working).
What should happen to yield? What happens if the opposite occurs?
Should go down. If it increases, the protein is membrane bound.
