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20.109(F22): Laboratory Fundamentals of Biological Engineering

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Two metrics will be used to evaluate the success of the protein purification protocol used to express and isolate PfFKBP35: 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 M2D2!) 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.

Sp17 20.109 M1D3 SDS-PAGE.png
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 PfFKBP35 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.


Part 1: Concentrate PfFKBP35 protein solution

Before evaluating the purity and concentration of the PfFKBP35 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. PfFKBP35 is ~35 kDa and the 6xHis-tag that was attached to the N-terminus for purification is ~2 kDa, therefore the total size of the purified protein is ~37 kDa.

  1. Retrieve the "elution" 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" 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 tube to the centrifugal filter unit.
    • Remove the cap from the filter unit, then pipette the purified PfFKBP35 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 PfFKBP35 protein that will be used for the BCA assay (Part 3) and the functional assay (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 PfFKBP35 protein that will be used for SDS-PAGE analysis.

Diagram of centrifugal filter and concentration protocol. (A) Centrifugal filter with 10 kDa cutoff is used to concentrate protein solution and remove contaminants. (B) Aliquots are collected at different steps of concentration procedure to assess protein expression and purity.

Part 2: Visualize PfFKBP35 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.

  1. Retrieve the aliquots that were collected during the protein purification and concentration procedures.
  2. To 30 μL of each aliquot, add 6 μL of Laemmli sample buffer.
    • Prepare the cell pellet by adding 30 μL of PBS 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 a molecular weight standard.
  6. Load the total volume for the samples and 10 μL of the ladder into the polyacrylamide 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.
  17. Image gel using the white light setting, not the UV light setting, of the gel documentation station.

In your laboratory notebook, complete the following:

  • For each lane of your polyacrylamide gel image provide the expected results.
    • Do you expect to see the PfFKBP35 protein in the lane?
    • Do you expect to see other cellular proteins in the lane?
  • When you receive the image of your polyacrylamide gel, fully interpret the data for every lane.
    • Do you see PfFKBP35 protein? Is the band the expected size?
    • Do you see other cellular proteins?
  • If there are unexpected results, give possible explanations.

Part 3: 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.

Prepare diluted bovine serum albumin (BSA) standards

  1. Obtain a 1 mL aliquot of 2.0 mg/mL BSA stock.
  2. Prepare your standards according to the table below using PBS as the diluent:
Volume of diluent (μL) Volume of BSA (μL) Final concentration of BSA (μg/mL)
A 0 300 of stock 2000
B 125 375 of stock 1500
C 325 325 of stock 1000
D 175 175 of B 750
E 325 325 of C 500
F 325 325 of E 250
G 325 325 of F 125
H 400 100 of G 25
I 400 0 0 = blank

Prepare Working Reagent (WR) and determine protein concentration

  1. You will create duplicate wells for each concentration of your standards as well as running your PfFKBP35 in duplicate.
  2. Use the following formula to calculate the volume of WR required: (# of standards + # unknowns) * 0.215 = total volume of WR (in mL).
  3. 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.
  4. Pipet 25 μL of each standard prepared in the previous section and 25 μL of your concentrated PfFKBP35 protein into separate, wells of a flat bottom 96-well plate.
  5. Add 200 μL of the WR to each 25 μL aliquot of the standard and to the PfFKBP35 concentrated protein.
    • The WR is added to each of the wells that was prepared in Step #3.
  6. Cover your plate with plastic wrap and incubate at 37 °C incubator for 30 min.
  7. Cool the plate to room temperature.
  8. Using a plate reader spectrophotometer, measure the A562 for each well.
  9. Next, use the numbers from the spectrophotometer to calculate the concentration of PfFKBP35.
  10. Subtract the A562 of the blank standard (I) from that of all the standard and PfFKBP35 samples.
    • This step subtracts the background 'noise' from the data measurements.
  11. Generate a standard curve by plotting the blank-corrected A562 for each BSA standard (A-I, B-I, ..., I-I) vs. its concentration in μg/mL.
  12. Use the standard curve to calculate the concentration of protein in the PfFKBP35 sample.

In your laboratory notebook, complete the following:

  • Graph a standard curve using the data collected from the BCA assay.
    • Include the equation of the trend line and R2 value.
  • What does the R2 indicate about the data that there were collected? Are you confident in these data? Why or why not?
  • Use the equation of the trendline to calculate the concentration of PfFKBP35 in protein solution.
    • Please note, the PfFKBP35 protein solution was diluted for use in the BCA assay.
  • 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 PfFKBP35 the only protein in the concentrated sample?

Reagents list

  • centrifugal filter unit, 10 kDa cutoff (from Millipore)
  • 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)
  • Precision Plus Dual Color Standard 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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