20.109(S20):Assess purity and concentration of TDP43 protein (Day3)

From Course Wiki
Jump to: navigation, search
20.109(S20): Laboratory Fundamentals of Biological Engineering

Sp20 banner image v2.png

Spring 2020 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Screening ligand binding        2. Measuring gene expression        3. Engineering antibodies              


Introduction

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.

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 on 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 determine the size and concentration of your purified TDP43-RRM12 protein. When electrophoresing uninduced and induced samples side-by-side, you should see a protein band at the expected molecular weight for TDP43-RRM12, which may be very faint or non-existent in the uninduced control sample, but bright and thick in the induced sample. 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. The molecular weight of TDP43-RRM12 is ~20 kDa. Each of the features adds 'weight' to the product: SnapTag is ~20 kDa, 6x-His and MBP are ~40 kDa. Critically think about what the expected band size is for each of the samples examined using SDS-PAGE. To visualize all the proteins released by the bacteria, you will stain the gels with Coomassie Brilliant Blue (actually, a variant called BioSafe Coomassie). Because this is a non-specific stain for all proteins, it will provide information concerning the purity of your protein sample.

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: BE Communication Lab workshop

Our communication instructors, Dr. Sean Clarke and Dr. Prerna Bhargava, will join us today for a workshop focused on writing a clear and impactful Abstract.

Part 2: Concentrate Snap-tag TDP43-RRM12 protein solution

To use the purified and Snap-tag TDP43-RRM12 protein, we will need to concentrate the amount of protein in solution (or eliminate the excess buffer). For this, we will use a centrifugal filter with a 3 kDa cutoff. The sample is first pipetted onto the filter, centrifuged to remove a fraction of fluid, and then recovered in a smaller volume using a pipet.

  1. Retrieve a 4 mL centrifugal filter unit from the front bench (it looks like a 15 mL conical tube with a filter unit).
  2. Remove the cap then pipette your purified Snap-tag TDP43-RRM12 solution into the filter unit and replace the cap.
    • Make sure to mark the tube with your team color.
  3. Give the filter unit to the teaching faculty. All the filter units for the lab section will be spun together in the centrifuge at 4500 g for 20 minutes.
  4. During centrifugation, prepare a 1.5 mL eppendorf tube, clearly labeled with your team name (a colored sticker with TR or WF) as well as the contents (Snap_TDP43-RRM12 concentrated).
  5. Once the centrifugation is complete, use a pipette to retrieve the remaining fluid on the filter and place in the labeled eppendorf tube.
    • Use a P200 pipet tip to ensure all the fluid is retrieved from the filter unit.
  6. Transfer 30 μL of the concentrated protein solution to a fresh eppendorf tube for Part #2.
  7. Make sure to give your final tube of concentrated protein to the Teaching Faculty. It will be stored at 4 °C until your next experiment.

Part 3: Visualize purified protein with polyacrylamide gel electrophoresis (PAGE)

During the previous laboratory session, you reserved an aliquot of your induced and the uninduced cell lysate and bacterial pellets. In addition, the flow-through from the wash steps was stored. Today you will use SDS-PAGE to visualize the effectiveness of IPTG/arabinose induction and the purification procedure.

  1. Retrieve the 30 μL aliquots of your induced (prepared by you on M1D2) and the uninduced (prepared by the Instructors) cell lysates. In addition you will need the bacterial pellet from the induced sample, the flow-through from the lysate (supernatent), the flow through from the first wash step, and the concentrated protein solution prepared in Part #1.
    • Transfer 30 μL from the flow-through from the lysate (supernatent) and the flow through from the first wash step into labeled 1.5 mL eppendorf tubes.
    • For the bacterial pellets add 30 μL of water to a labeled 1.5 mL eppendorf tube, dip a clean pipette tip in the bacterial pellet then swirl into the water, transferring a small amount of bacteria.
  2. Add 6 μL of Laemmli sample buffer to all of the samples prepared for SDS-PAGE. There should be 6 tubes total.
  3. Boil all samples for 5 min in the water bath located in the chemical fume hood. If that water bath is full, use the dry bath located on the front bench.
    • Secure the caps with the cap-locks located in the fume hood to ensure that the eppendorf caps do not pop open during the boiling step as this will result in your sample escaping the tube.
    • Spin your samples for 30 sec before loading.
  4. 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.
  5. Record the order in which you will load your samples and molecular weight standards in the polyacrylamide gel.
  6. When you are ready to load your samples, alert the teaching faculty.
    • Please watch the demonstration closely to ensure your samples are correctly loaded and the polyacrylamide gel is not damaged during loading.
  7. Your samples will be electrophoresed at 200 V for 30-45 min.
  8. Following electrophoresis, alert the teaching faculty who will use a spatula to carefully pry apart the plates that encase your polyacrylamide gel.
  9. Using wet gloves, transfer your 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. Empty 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 for 60 min at room temperature on the rotating table(or overnight depending on time).
  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.
  16. Wash the gel for the remainder of the class on the rotating table.
    • Replace the dH2O every 30 min.

Tomorrow the teaching faculty will transfer your gel to fresh dH2O and take a photograph. The image will be posted to the Class Data page.

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.

Part 4a: Prepare diluted albumin (BSA) standards

  1. Obtain a 100 μL aliquot of 2.0 mg/mL albumin standard stock and a conical tube of PBS from the front bench.
  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

Part 4b: 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.
    • Prepare your WR in a 1.5ml microcentrifuge tube.
  3. Pipet 25 μL of each standard prepared in Part 4a and 25 μL of your purified 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 OR TDP43-RRM12 protein sample (do not mix the standard and TDP43-RRM12).
  5. Cover your plate with plastic wrap and incubate at 37°C incubator in the back of the lab for 30 min.
    • During the incubation step, check on your SDS-PAGE gel.
  6. Cool the plate to room temperature.
  7. Notify the teaching faculty you need to take your samples to the spectrophotometer plate reader.
    • Obtain the absorbance at 562 nm for each well. 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.
    • 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.
    • Use the standard curve to determine the protein concentration of purified TDP43-RRM12 in your sample.

Reagents

  • SDS-PAGE Gels, buffer and ladder from Bio-Rad
    • 4-20% polyacrylamide gels in Tris-HCl
    • TGS buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS, pH 8.3)
    • Dual color Standard ladder and unstained ladder
    • Unstained marker masses in manual linked here
  • BioSafe Coomassie G-250 Stain (Bio-Rad)
  • Pierce BCA protein assay (ThermoFisher)
  • 6x Reducing Laemmli Sample Buffer (Boston BioProducts)

Navigation links

Next day: Perform small molecule microarray (SMM) with TDP43 protein

Previous day: Purify TDP43 protein