Difference between revisions of "20.109(S22):M1D4"

Introduction

Two metrics will be used to evaluate the success of the protein purification protocol used to express and isolate TDP43-RRM12: 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 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 TDP43-RRM12 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: Participate in Communication Lab workshop

Our communication instructor, Dr. Prerna Bhargava, will join us today for a discussion on crafting data figures and legends.

Part 2: Visualize TDP43-RRM12 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.

One important note to consider is that the expected size of the TDP43-RRM12 product is dependent on the features that were added during the cloning process (review your notes from M2D1 prelab discussion!). The molecular weight of TDP43-RRM12 is ~20 kDa. Each of the features adds 'weight' to the product: SnapTag is ~20 kDa, 3C recognition site is ~1 kDa, MBP is ~40 kDa, and 6x-His is ~1 kDa. As you evaluate each lane of the SDS-PAGE results below, critically think about what the expected band size is for each of the samples. Review the M1D3 prelab slides for the orientation of the components that makeup the purified product.

To see a closer view of loading protein sample into an SDS-PAGE gel, please watch the video linked here: [Loading samples into a protein gel].

1. Retrieve the aliquots that were collected during the protein purification and concentration procedures.
   • In addition to the aliquots that were prepared in the previous laboratory session, you will also include the desalted protein in the SDS-PAGE experiment. To do this, transfer 30 μL from the 15 mL conical with your desalted protein into a microcentrifuge tube.
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 (set at 100 °C) located at the front laboratory bench.
   • Secure the tubes with the cap-locks located next to baths 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. Vortex and centrifuge samples for 15 seconds each to resuspend and collect samples in the bottoms of the tubes, respectively. Vortex and table-top mini centrifuge are at front bench.
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. Cover the gel with the BioSafe Coomassie in 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:

• Consider the expected results for each lane in the polyacrylamide gel.
  • Do you expect to see the TDP43-RRM12 protein in the lane? If yes, what is the expected size? If no, why not?
  • Do you expect to see other cellular proteins in the lane? If yes, why? If no, why not?

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.

To ensure the steps included below are clear, please watch the video tutorial linked here: [BCA assay].

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:

Tube	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. Use the following formula to calculate the volume of WR required: (# of standards + # unknowns) * 0.215 = total volume of WR (in mL). #Standards and unknowns will be run in duplicate.
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 TDP43-RRM12 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 TDP43-RRM12 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 A₅₆₂ for each well.
8. Subtract the A₅₆₂ of the blank standard (I) from that of all the standard and TDP43-RRM12 samples.
   • This step subtracts the background 'noise' from the data measurements.
9. 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.
10. Use the standard curve to calculate the concentration of protein in the TDP43-RRM12 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 TDP43-RRM12 in protein solution.
• 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 TDP43-RRM12 the only protein present in the concentrated sample? How do you know?

Reagents list

• 6x Reducing Laemmli Sample Buffer (from Boston BioProducts)
• 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)

Navigation links

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