20.109(S16):Characterize protein expression (Day7)

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20.109(S16): Laboratory Fundamentals of Biological Engineering

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Introduction

Today you will obtain titration curves against calcium for your wild-type and mutant proteins using an automated fluorescence plate reader. This machine reads multiple samples in a standard format – in our case, a 96-well microtiter plate. The output is a grid of up to 96 fluorescence values, for rows A-H and columns 1-12, which is amenable to analysis with a program like Excel.

Signal:noise in arbitrary data collection. Background measurements (open circles), sample measurements (closed circles), and average values (short horizontal lines) are shown. The short line without any data points represents the reduction in average signal when background is subtracted. All measurements are with respect to an arbitrary vertical axis; the long horizontal line represents a measurement of zero.

While the concept of scale is a pragmatic concern, a perhaps more substantive topic of interest to us today is that of confidence in our results. As you are probably well-aware, every manipulation and measurement you make in the lab has an error associated with it. For example, consider the ubiquitous P200 pipetman. According to one pipet manufacturer, its accuracy is 1% (slightly worse at the lowest volumes). So an attempt to pipet 100 μL would result in an actual volume of 99-101 μL from the error of the instrument alone, which could be further compounded by a sleepy pipet operator, say. The precision of a pipet is typically better than its accuracy, 0.25% for the example given above. Precision refers to the reproducibility of a given measurement, not its absolute accuracy. This simple example demonstrates the general principles applicable to other types of error.

You will attempt to get a sense of the overall error of today’s experiment by running your protein samples in duplicate. That is, for each protein-calcium combination, you will perform two independent measurements. These measurements can then be averaged to smooth out your data, and hopefully improve the signal to noise ratio - where signal here refers to true differences between samples mixed with different amounts of calcium, and noise means inherent fluctuations in the system due to error. Noise in this experiment can also refer to background fluorescence of the sample buffer. Thus, another way to maintain a reasonable signal:noise ratio is by keeping our protein fairly concentrated, so that the absolute fluorescence values we obtain are high compared to the background. The figure at right demonstrates the above concepts. Scatter in the data (not all of the circles are at the same height) is one kind of noise. The level of background is another kind of noise: the left-hand data has a relatively low signal and thus poor signal:noise ratio, while the right-hand data has a relatively high absolute signal and improved signal:noise ratio.

Regarding the assay itself, note that we are using specially paired buffers to keep the concentration of calcium constant inside a given well. Otherwise, at low concentrations, as calcium began to bind to IPC it would be lost from the solution, thus distorting our measurement of binding and ultimately Kd.

Protocols

Everyone will begin with Part 1, due to its lengthy incubation steps. After that, about two groups at a time will work on Part 2.

Part 1: SDS-PAGE

  1. Last time you prepared cell-normalized -IPTG and +IPTG samples and added Laemmli sample buffer (containing SDS, etc) to them. You also have aliquots of your purified wild-type and X#Z mutant IPC proteins. Now you will complete protein denaturing in preparation for PAGE, alongside two different MW ladders.
    • The pre-stained ladder will be used to track gel progress.
    • The unstained ladder contains a known amount of protein per band and thus can be used to estimate gross protein contents.
  2. Boil all 6 samples for 5 minutes in the water bath located in the fume hood.
    • Be sure to put cap-locks on the eppendorf tubes that contain your samples to ensure the lids do not open during boiling.
  3. You will be shown by the teaching faculty how to load your samples into the SDS-PAGE gel. You should load your samples according instructions below, one team per gel.
  4. Note the starting and stopping time of electrophoresis, which will be initiated by the teaching faculty at 200 V, and run for 30-45 minutes.
  5. Pry apart the plates using a spatula, and carefully transfer your gel to a staining box.
  6. Add enough distilled water to cover the gel (~200 mL) and rinse the gel for 5 minutes on the shaker.
  7. Repeat the rinse two more times with fresh water (~200 mL and 5-minute incubation each time).
  8. Add 50 mL of BioSafe Coomassie, and incubate for at least 1 hour.
  9. Empty the staining solution into the waste container in the fume hood - careful not to lose your gel!
  10. Add 200 mL of water to your stained gel. Replace with fresh water just before leaving the lab if you have a chance.
  11. Tomorrow, the teaching staff will transfer each gel to fresh water, then photograph them and post the results to the Day 7 Discussion page.

Loading order and volumes

Loading volume is 10 μL for the ladder and 18 μL for your samples.

You may load your samples in any order you choose. Keep a few things in mind as you decide on the order:

  • Record the order in your notebook!
  • Think about how you will want to discuss your data in the Protein engineering summary. This will likely be a figure in your report and the order of your samples can help you logically report and describe your data.
  • You have 8 samples total: stained ladder, unstained ladder, WT cell lysate before and after IPTG induction, X#Z mutation cell lysate before and after IPTG induction, purified WT protein, and purified X#Z mutated protein.

It's best to not use well 1 and 10, because the edge lanes on an SDS-PAGE often appear somewhat distorted.

Part 2: Prepare samples for titration curve

Tips for success

Take great care today to limit the introduction of bubbles in your samples. When expelling fluid, pipet slowly while touching the pipet tip against the bottom or side of the well.

Protocol

Titration plate map
  1. Take a black 96-well plate, and familiarize yourself with the plate map scheme at right: top two rows are to be loaded with wild-type IPC, next two rows are to be loaded with your X#Z mutant IPC, and the final row is to be loaded with water/BSA to serve as a blank/background row.
    • The dark sides of the plate reduce "cross-talk" (i.e., light leakage) between samples in adjacent wells, another potential contribution to error.
  2. Aliquot your wild-type protein to your plate. Use your P200 pipet to add 30 μL of protein (per well) to rows A and B of your plate.
  3. Aliquot you X#Z mutant IPC to your plate. Use your P200 pipet and add 30 μL of protein (per well) to rows C and D of your plate.
  4. Finally, add 30 μL of water with only 0.1% BSA (no IPC) to row 5(E) of your plate.
  5. The calcium solutions are at the front bench in shared reservoirs. Carefully carry your plate to the front bench to add these solutions with the multi-channel pipet.
  6. Using shared reservoir #1 (lowest calcium concentration - actually 0 nM), add 30 μL to the top five rows in the first column of the plate. Discard the pipet tips.
  7. Now work your way from reservoirs #2 to #12 (highest calcium concentration), and from the left-hand to the right-hand columns on your plate. Be sure to use fresh pipet tips each time! If you do contaminate a solution, let the teaching faculty know so they can put out some fresh solution. Honesty about a mistake is far preferred here to affecting every downstream experiment.
  8. When you are done, alert the teaching faculty and you will be taken in small groups to measure the fluorescence values for your samples.

Part 3: Fluorescence assay

  1. The BMC (BioMicro Center) has graciously agreed to let us use their plate reader. Walk over to building 68 with a member of the teaching staff.
  2. You will be shown how to set the excitation (485 nm) and emission (515 nm) wavelength on the plate reader to assay your protein.
  3. Your raw data will be posted on today's Discussion page and emailed to you as a .txt file so you can begin your analysis.

Reagent list

  • Gels from Bio-Rad
    • 4-15% polyacrylamide gels in Tris-HCl
    • TGS buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS, pH 8.3)
    • Kaleidoscope and unstained marker sizes here
    • Unstained marker masses in manual linked here
  • Calcium calibration kit from Life Technologies
    • Zero free calcium buffer: 10 mM EGTA in 100 mM KCl, 30 mM MOPS, pH 7.2
    • 39 μM free calcium buffer: 10 mM CaEGTA in 100 mM KCl, 30 mM MOPS, pH 7.2
  • Thermo Scientific Varioskan Flash Spectral Scanning Multimode Reader

Navigation links

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