Download During the last lab session you grew a culture of E

2/20/2016 – Biotech 3
Name_____________________________
Lab 10
Affinity Column Chromatography
During the last lab session you grew a culture of E. coli JM101 that had been transformed with a pQE9
vector which has an insert for the C-terminal domain of the NarL protein (NarLC, molecular weight =
9,615 Da). When the culture reached an OD600 0.4-0.8 (i.e. when the culture was in the exponential
growth phase), IPTG was added to a final concentration of 2 mM to overexpress the NarLC protein. At
the end of the overexpression, you harvested the bacteria by centrifuging the culture for 5 minutes at 3000
rpm; obtaining a large bacterial pellet that was then frozen at -80 °C and saved for today’s lab. In today’s
lab you will lyse your E. coli cells and purify the overexpressed NarLC protein.
Preparation
1. First calculate how to make each stock solution. Each group must show their calculations before
proceeding to the next step.
2. Next, prepare 50 ml of each buffer as listed below using the available stock solutions.
3. Calculate the volume of each stock solution that you will need to prepare each buffer. Note:
Imidazole was not prepared as a stock solution and will need to be weighed out and added to the wash
and elution buffers. The molecular weight of Imidazole is 68.08 g/mol. All calculations need to be
entered into your lab manual. Titrate your buffers using the pH meter to the appropriate pH with HCl
or NaOH as needed. Make sure to wear safety goggles, gloves, and a lab coat!
Available Stock Solutions
1 M Tris
50 % Glycerol
10 % Triton X-100
5 M NaCl
1M MgCl2
Lysis Buffer (50 ml)
50 mM Tris pH 8.0
0.1% Triton X-100
10% Glycerol
2 mM MgCl2
1X DNase (do not add)
1X Lysozyme (do not add)
0.5 M Dibasic Sodium Phosphate
0.5 M Monobasic Sodium Phosphate
1000X DNase (10 mg/ml)
1000X Lysozyme (100 mg/ml)
Binding Buffer (50 ml)
50 mM NaPhosphate pH 7.8
10 mM Imidazole
0.1 M NaCl
1% Glycerol
1 mM MgCl2
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Elution Buffer (50 ml)
50 mM NaPhosphate pH 7.8
0.4 M Imidazole
0.1 M NaCl
1% Glycerol
1 mM MgCl2
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Cell Lysis
1. Resuspend the frozen pellet in 5 ml of lysis buffer. Note: Protein degradation is undesirable when
purifying a protein. Therefore, lysis buffer generally contains a protease inhibitor such as PMSF in
order to prevent protein degradation. Protease inhibitors are typically very expensive, and may be
extremely toxic as in the case of PMSF. We will avoid using protease inhibitors in this laboratory.
Luckily the protein we are purifying (NarLC) is not quickly degraded by proteases.
2. Add DNase and Lysozyme to the 5 ml resuspended culture to a final concentration of 1X.
3. Incubate the resuspended culture on ice for 30 minutes.
4. Homogenize the resuspended culture using the piston homogenizer as demonstrated by the instructor.
The lysed cell suspension is referred to as the cell lysate.
5. Centrifuge for 20 minutes at 5000 rpm at 4 oC. Typically this step is performed at a higher rpm value
(between 12,000 – 40,000 rpm). The Sorvall centrifuge in the laboratory does not go up to that high
rpm. DO NOT attempt to set the centrifuge to a higher rpm value than what the rotor is rated for. To
do so is very dangerous and can severely injure someone or worse.
6. After the spin cycle, collect the supernatant into a new tube. If the supernatant is still viscous add an
additional 5 μl of DNase and 5 μl of 1 M MgCl2. The supernatant is referred to as the cell extract
and contains our protein of interest since it is a soluble protein. The pellet contains cell debris and
unlysed cells. Note: if our protein of interest was membrane bound or present in inclusion bodies
(misfolded and aggregated protein), then our protein would be located in the pellet.
7. Prepare the affinity column for protein purification.
Note the importance of each reagent in the Lysis Buffer:
a) 50mM Tris pH 8.0 – maintains desired pH
b) 10% glycerol – maintains protein stability and prevents protein aggregation. However, glycerol
may pose a problem in subsequent experiments such as in Nuclear Magnetic Resonance (NMR)
used to determine protein structure.
c) 0.1% Triton X-100 – Detergent. Prevents aggregation of hydrophobic and membrane proteins.
Detergents chosen for the lysis solution should be specific to the proteins.
d) 100 μg/ml lysozyme – Degrades bacterial cell wall. Lysozyme MW is approximately 15Kd. If
studying the induction of a protein with a similar MW, you may want to consider avoiding
lysozyme.
e) 1mM PMSF and /or more anti-proteases – Prevents protein degradation. EDTA is a good
alternative but must be avoided when using chelating purification resin as in today’s lab.
f) 100 μg/ml DNase + 1 mM Mg Cl2 – Degrades DNA, MgCl2 is a cofactor in DNase.
Affinity Column Procedure
1. Obtain an aliquot of affinity chelating resin from the instructor.
2. Load the entire volume of resin onto the provided column.
3. The resin is stored in a 20% ethanol solution and 0.1 mM EDTA which needs to be washed off.
Wash the resin three times with 5 ml of water.
4. Load 1 ml of 0.5 M NiSO4. The resin will bind to the dissolved Ni2+ and will turn the resin green.
Do not use serological pipets for column chromatography. Either use a pipetman, or better yet use
disposable transfer pipette.
5. Wash off excess Ni2+ three times with 5 ml of water.
6. Wash the resin with 1 ml of Elution buffer. This step elutes off anything that may have been bound to
the resin.
7. Wash the resin with 5 ml of water. This step washes away the high imidazole from the previous
wash.
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8.
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Wash the resin with 5 ml of Binding Buffer. This step prepares the column with a buffer that is
optimal for protein binding.
Save 100 μl the cell extract in a microfuge tube (this will be the Cell Extract sample). Load the rest
of the cell extract onto the column. Collect all of the flow through in a 15 ml conical tube. The flow
through is comprised of proteins that do not bind to the resin, including NarLC that did not bind due to
capacity constraints.
Wash the resin with 10 ml of additional binding buffer. Collect two 250 μl fractions and then
subsequent 500 μl fractions!! The first two fractions will be the Wash samples.
Elute NarLC by washing the column with 5 ml of Elution buffer. Collect three 250 μl fractions and
then subsequent 500 μl fractions!! The first three fractions will be the Elution samples.
After you are finished collecting the Eluted NarLC protein, wash the column with an additional 5 ml
of Elution buffer. This step ensures that the majority of protein is eluted off.
Recharge the column by washing with 2 ml 0.25 M EDTA. Ni2+ is a divalent cation, and as you
know EDTA chelates divalent cations. Hence, why EDTA is avoided in the Binding and Elution
buffers. If EDTA were present in the buffers, Ni2+ would be stripped from the column, taking our
protein with it.
Perform a final wash with 5 ml of 20% Ethanol.
DO NOT THROW AWAY THE RESIN. Resuspend the resin in 5 ml of 20% Ethanol and place
the resin back into the original 5 ml conical tube.
Sample Preparation for SDS-PAGE
You will analyze your protein overexpression and purification by performing an SDS-PAGE.
1. During the last lab session, you prepared a “before induction” as well as an “after induction” cell
samples by collecting and centrifuging 1 ml of cell culture before the addition of IPTG and 1 ml of
cell culture at the time harvest. The pellet was frozen and stored at -80 °C. Also, the OD600 of the
before and after induction culture was recorded.
2. Resuspend the frozen pellets in 1X SDS loading buffer. Use 100 μl of 1X SDS loading buffer for
every 0.1 OD600. These will be the Before Induction and After Induction samples.
3. Next prepare the collected samples obtained during the NarLC purification.
4. Add 33 μl of 4X Laemmli SDS sample buffer to 100 μl of each sample collected from the column;
Cell Extract, Wash, and Elution samples.
5. Heat all of the samples for 10 minutes at 90 °C.
6. Load 10 μl of the Kaleidoscope molecular weight marker into the first well.
7. Load 15 μl of each sample into its own well.
8. Run the gel at 120 V for 1 hour or until the lower dye reaches the bottom of the gel.
9. Remove the gel from the running apparatus
10. Break open the plastic gel compartment to expose the gel.
11. Transfer the gel to staining dish containing protein stain solution.
12. Stain and destain the gel as instructed in the previous SDS-PAGE experiment.
13. Save the protein stain reagent after staining.
14. Take a picture of the stained gel.
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Post lab:
Did the protein expression work? How can you tell?
Plot the molecular weight vs. distance migration for the protein standard using the provided graph paper.
Remember to take the Log10 of the molecular weight before plotting your results.
Calculate the size of NarLC.
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