Difference between revisions of "20.109(F20):M2D3"

Introduction

Two metrics will be used to evaluate the success of the protein purification protocol used to express and isolate PF3D7_1351100: purity and concentration.

Protein purity
Electrophoresis is a technique that separates large molecules by size using an applied electrical field and a sieving matrix. DNA, RNA and proteins are the molecules most often studied with this technique; agarose (review M2D1!) and acrylamide gels are the two most common sieves. The molecules to be separated enter the matrix through a well at one end and are pulled through the matrix when a current is applied across it. The larger molecules get entwined in the matrix and are stalled; the smaller molecules wind through the matrix more easily and travel farther away from the well. The distance a nucleic acid or amino acid fragment travels is inversely proportional to the log of its length. Over time fragments of similar length accumulate into “bands” in the gel.

You will use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate your purified protein. SDS is an ionic surfactant, or detergent, which denatures the proteins and coats them with a negative charge. Since denatured proteins are linear, they will move through the gel at a speed inversely proportional to their molecular weight, just like DNA in agarose gels. Non-denatured proteins run according to their molecular weight, shape, and charge. You will electrophorese a reference ladder containing proteins of known molecular weight and amount to evaluate the purity of the PF3D7_1351100 protein. The purity of the protein is assessed by visualizing the number of protein bands in the sample. If only a single band consistent with the expected size of the purified protein is present, the sample is pure because only the protein of interest is present. If additional bands are present, additional proteins are present. Depending on the experiments that will be completed with the protein, it is more / less important to have pure protein. In addition to purity, the efficiency of protein expression is visualized using SDS-PAGE. The protein purification procedure is completed with both an uninduced cell culture and an induced cell culture. When these samples are electrophoresed, you should see a protein band at the expected molecular weight for the protein of interest in the induced sample. For the uninduced sample, the band should be absent or very faint. To visualize the protein bands in the polyacrylamide gel, Coomassie Brilliant Blue stain (actually, a variant called BioSafe Coomassie) will be used. Because this is a non-specific stain for all proteins it will provide information concerning the purity of your protein sample.

Protein concentration
To measure the concentration of your purified protein, you will use the BCA Protein Assay Reagent Kit. This kit enables colorimetric detection and quantification of the total protein within a sample. The ability to measure protein concentration is based on the detection of Cu¹⁺ by the detection reagent, bicinchoninic acid (BCA). The Cu¹⁺ is formed when Cu²⁺ is reduced by protein in an alkaline environment. Through this reduction reaction, a purple product is formed by the chelation of BCA and Cu¹⁺ at a 2:1 ratio. This water-soluble complex has a strong absorbance at 562 nm. Because absorbance and protein concentration have a linear relationship, it is possible to compare the absorbance of an unknown protein sample to a standard curve, generated with samples of known protein concentrations, and calculate the concentration of protein in the experimental sample.

Protocols

Part 1: Concentrate PF3D7_1351100 protein solution

Before evaluating the purity and concentration of the PF3D7_1351100 protein, it is important to concentrate the protein solution (the protein solution is elution 1 from the previous laboratory session!). Concentrating the protein eliminates excess buffer and contaminants. To do this, a centrifugal filter with a 10 kDa cutoff will be used. The cutoff value refers to the size of the molecules that are able to pass through the filter -- molecules smaller than 10 kDa flowthrough the filter whereas molecules larger than 10 kDa are retained in the reservoir above the filter. One critical consideration when selecting a centrifugal filter is the molecular weight of the protein of interest. The protein should be larger than the cutoff value to ensure it is retained in the reservoir above the filter. PF3D7_1351100 is ~17.5 kDa and the 6xHis-tag that was attached to the N-terminal for purification is 2.5 kDa, therefore the total size of the purified protein is ~20 kDa.

1. Retrieve the "elution 1" sample you collected in the previous laboratory session.
2. Aliquot 30 μL of the elution 1 to a fresh microcentrifuge tube.
   • Label the microcentrifuge tube containing the aliquot as "elution 1" and give it to the Instructor! This aliquot will be used later when protein expression and purity are examined.
3. Add the remaining contents of the original elution 1 tube to the centrifugal filter unit.
   • Remove the cap from the filter unit, then pipette the purified PF3D7_1351100 protein solution into the filter unit and replace the cap.
4. Centrifuge at 4500 g for 20 minutes.
5. After centrifugation, transfer the liquid from the top of the filter unit to a fresh microcentrifuge tube.
   • Label the microcentrifuge tube containing the liquid as "concentrated protein" and give it to the Instructor! This aliquot will be used later when protein expression and purity are examined.
   • This tube contains the purified and concentrated PF3D7_1351100 protein that will be used for the BCA assay (below) and the SMM experiment (next laboratory session).
6. From the "concentrated protein" tube, aliquot 30 μL to a fresh microcentrifuge tube.
   • Label the microcentrifuge tube containing the concentrated protein as "concentrated protein for gel" and give it to the Instructor! This aliquot will be used later when protein expression and purity are examined.
   • This tube contains an aliquot of the purified and concentrated PF3D7_1351100 protein that will be used for SDS-PAGE analysis.

Part 2: Visualize PF3D7_1351100 protein using polyacrylamide gel electrophoresis (PAGE)

As discussed above, SDS-PAGE is used to evaluate the purity protein. It is also possible to track the purification process using SDS-PAGE. For this, several aliquots were collected during the protein purification and concentration procedures. Each of these aliquots will be electrophoresed and the presence / absence of the protein of interest in each of the aliquots will provide information on the success of the purification.

To ensure the steps required for polyacrylamide gel electrophoresis, the Instructor will provide a live demonstration of this process. You should include notes regarding the procedure in your laboratory notebook!

1. Retrieve the aliquots that were collected during the protein purification and concentration procedures.
   • In addition to the aliquots that were collected from the IPTG-induced Nico(DE3) pET-28b(+)_PF3D7_1351100 cell culture, a cell lysate aliquot was collected from an uninduced cell culture.
2. To 30 μL of each aliquot, add 6 μL of Laemmli sample buffer.
   • Prepare the cell pellet by adding 30 μL of water to a fresh microcentrifuge tube, then dip a clean pipette tip in the bacterial pellet and swirl into the water. Lastly, add 6 μL of Laemmli sample buffer.
3. Boil all samples for 5 min in the water bath located in the chemical fume hood.
   • Secure the tubes with the cap-locks located in the fume hood to ensure that the caps do not pop open during the boiling step as this will result in your sample evaporating from the tube.
4. Centrifuge samples to collect samples in the bottoms of the tubes.
5. You will load all samples and two molecular weight standards.
   • A pre-stained ladder will be used to track the migration of your samples through the polyacrylamide gel.
   • An unstained ladder with bands of known amounts of protein will be used to estimate protein concentration in your samples.
6. Load the samples and ladders into the polyacrylamid gel.
7. Electrophorese at 200 V for 30-45 min.
8. Following electrophoresis, remove the cartridge from the electrophoresis chamber and carefully pry apart the plastic plates that encase the polyacrylamide gel.
9. With wet gloves, transfer the polyacrylamide gel to a staining box and add enough dH₂O to cover the gel.
10. Wash the gel for 5 min at room temperature on the rotating table.
11. Decant the water from the staining box in the sink.
    • Be careful that the gel does not fall into the sink!
12. Repeat Steps #10-11 a total of 3 times.
13. Add 50 mL of BioSafe Coomassie to the staining box and incubate on the rotating table at room temperature overnight.
14. Empty the BioSafe Coomassie into the appropriate waste container in the chemical fume hood.
    • Be careful that the gel does not fall into the waste container!
15. Add 200 mL of dH₂O to the staining box to wash the excess stain from the gel.
16. Incubate on the rotating table at room temperature for 2 hrs.
    • Replace the dH₂O every 30 min.

In your laboratory notebook, complete the following:

• For each well in the polyacrylamide gel image provided by the Instructors, fully interpret the data.
  • Do you expect to see the PF3D7_1351100 protein in the lane? Do you see PF3D7_1351100 protein? Is the band the expected size?
  • Do you expect to see other cellular proteins in the lane? Do you see other cellular proteins?
• If there are unexpected results, give possible explanations.

Part 4: Measure protein concentration

SDS-PAGE enables you to visualize the presence of protein and provides information concerning the purity of your protein sample(s). Though comparing SDS-PAGE band intensities between samples and molecular weight standards gives an estimate of protein concentration, using a standard curve generated from samples of known protein concentration is a more precise method for measuring protein concentration.

To ensure the steps included below are clear, please watch the video tutorial (linked [[ | here]]). The steps are detailed below so you can follow along!

Prepare diluted albumin (BSA) standards

1. Obtain a 100 μL aliquot of 2.0 mg/mL albumin standard stock.
2. Prepare your standards according to the table below using PBS as the diluent:

Vial	Volume of diluent (μL)	Volume (μL) and source of albumin (vial)	Final albumin concentration (μg/mL)
A	700	100 of stock	250
B	400	400 of A	125
C	450	300 of B	50
D	400	400 of C	25
E	400	100 of D	5
F	400	0	0 = blank

Prepare Working Reagent (WR) and measuring protein concentration

1. Use the following formula to calculate the volume of WR required: (# of standards + # unknowns) * 0.215 = total volume of WR (in mL).
2. Prepare the calculated volume of WR by mixing 50 parts of BCA Reagent A with 1 part BCA Reagent B (50:1 or 50-fold dilution of B).
   • For example, if your calculated total volume of WR is 100 mL, then mix 98 mL of A, and 2 mL of B.
3. Pipet 25 μL of each standard prepared in the previous section and 25 μL of your concentrated PF3D7_1351100 protein into separate, wells of a flat bottom 96-well plate.
4. Add 200 μL of the WR to each 25 μL aliquot of the standard and to the PF3D7_1351100 concentrated protein.
   • The WR is added to each of the wells that was prepared in Step #3.
5. Cover your plate with plastic wrap and incubate at 37 °C incubator for 30 min.
6. Cool the plate to room temperature.
7. Using a plate reader spectrophotometer, measure the absorbance at 562 nm for each well.
8. Subtract the measurement of the Blank standard (F, after adding working reagent and being incubated) from that of all other individual standard and unknown samples.
   • This accounts for the background absorbance.
9. Generate your standard curve by plotting the Blank-corrected absorbance 562 for each albumin standard (A-F, B-F, ..., F-F) vs. its concentration in μg/mL.
10. Use the standard curve to calculate the concentration of protein.

In your laboratory notebook, complete the following:

• Why is it useful to evaluate the purity and measure the concentration of protein that will be used in experiments?
  • Hint: consider the specificity of each technique and what information is provided by each method. Is PF3D7_1351100 the only protein in the concentrated sample?

Reagents list

• centrifugal filter unit, 10 kDa cutoff (from Millipore)
• SDS-PAGE Gels, buffer and ladder from Bio-Rad
• 4-20% polyacrylamide gels in Tris-HCl (from Bio-Rad)
• TGS buffer: 5 mM Tris, 192 mM glycine, 0.1% (w/v) SDS (pH 8.3) (from Bio-Rad)
• Dual color Standard ladder (from Bio-Rad)
• Unstained ladder (from Bio-Rad)
  • Molecular weights of ladder bands (linked here).
• BioSafe Coomassie G-250 Stain (from Bio-Rad)
• Pierce BCA protein assay (from ThermoFisher)
• 6x Reducing Laemmli Sample Buffer (from Boston BioProducts)

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