Materials and Reagents

Biological materials

Twelve-day-old Arabidopsis thaliana ecotype Columbia (Col_0) seedlings

Solutions

DEPC-treated water
Milli-Q water treated with 0.2% (v/v) DEPC for 24 h at room temperature and autoclaved for 20 min in a glass bottle. Cool down the bottled water at room temperature for 24 h before use. (Can be stored indefinitely if the bottle is unopened)
All reagent stocks (except sucrose solution, cycloheximide, RNase inhibitor and DTT) should be made in a glass beaker and stored in a 100 mL glass bottle in the dark at room temperature. (Replace with new stock after three months)
Cycloheximide (CHX, 50 mg/mL) stock is made by resuspending 50 mg of cycloheximide in 1 mL of ethanol in a 1.5 mL tube. Store stock solution at -20 °C. (Replace with new stock after three months)
Chloramphenicol (CHL, 50 mg/mL) stock is made by resuspending 50 mg of chloramphenicol in 1 mL of ethanol in a 1.5 mL tube. Store stock solution at -20 °C. (Replace with new stock after three months)
Dithiothreitol (DTT, 1 M) stock is made by resuspending 0.154 g of DTT in 1 mL of water in a 1.5 mL tube. Store stock solution at -20 °C. (Replace with new stock after two months)
Sucrose solutions made in a glass beaker need to be filtered through a 0.2 µm PVDF filter bottle and stored in the dark at 4 °C. (Replace with new stock after one month. Before each use, always shake the bottle to check for any cloudy appearance. Discard and make new solutions as necessary)
Punch solution should be made in a glass bottle and autoclaved for 20 min on liquid cycle. (Can be stored indefinitely at room temperature in the dark)
100 mL of 1 M Tris-HCl (pH 8.4) is made by dissolving 12.14 g of Tris in 80 mL of deionized water and adjusting the pH to 8.4 using 0.1 N HCl. After attaining the desired pH, the volume of 1 M Tris-HCl solution is adjusted to final 100 mL using deionized water. (Solution is stored at room temperature and it is recommended to replace with new stock after three months)
Lysis buffer (1,000 µL in a 2 mL tube) (see Recipes)
15% (or 50%) sucrose solution (100 mL) (see Recipes)
Punch solution (100 mL) (see Recipes)

Recipes

Lysis buffer (1,000 µL in a 2 mL tube)

Make fresh from stock solutions for each experiment.

Reagent

Final concentration

Amount

Tris-HCl (1 M, pH 8.4)

KCl (0.5 M)

MgCl₂ (0.5 M)

Deoxycholic acid (10%)

Polyoxyethylene 10 tridecyl ether [20% (w/v)]

Cycloheximide (50 mg/mL)

RNase inhibitor (40 U/µL)

H₂O

200 mM

50 mM

25 mM

1% (v/v)

2% (v/v)

50 µg/mL

40 U/mL

n/a

200 µL

50 µL

25 µL

100 µL

100 µL

1 µL

1 µL

523 µL

Total

n/a

1,000 µL

15% (or 50%) sucrose solution (100 mL)

Reagent Final concentration Amount
Tris-HCl (1 M, pH 8.4)
KCl (0.5 M)
MgCl₂ (0.5 M)
Sucrose
H₂O
200 mM
50 mM
25 mM
15% or 50% (w/v)
n/a
20 mL
10 mL
5 mL
15 or 50 g
40 mL
Total n/a Make up final volume to 100 mL

Punch solution (100 mL)
*Note: Water should be added in increments of 10 mL. Allow the solution to mix completely before adding any more water. Sucrose will take a substantial volume when fully dissolved.

Reagent Final concentration Amount
Sucrose
Bromophenol blue (0.1% (w/v))
Tris-HCl (1 M, pH 8.4)
H₂O
60% (w/v)
0.02% (v/v)
1 mM
n/a
60 g
20 mL
100 µL
see note*
Total n/a 100 mL

Laboratory Supplies

1.5 and 2 mL tubes (Thermo Fisher, catalog numbers: AM12450 and AM12475)
Pipettes (200–1,000, 20–200, 2–20, and 0.2–2 µL)
DNase/RNase free tips (1,000, 200, 10 µL)
1.5 mL tube rack
7/16 × 13/8 in. (11 × 34 mm) 2.2 mL polypropylene tube (Beckman Coulter, catalog number: 347357)
19 Gauge needle for gradient tube piercing (Fisher Scientific, catalog number: 14-826-52)
Parafilm (Fisher Scientific, catalog number: S37440)
Disposable bottle top filters (Thermo Fisher, catalog number: 597-4520)
Disposable bottle filter units (Thermo Fisher, catalog number: 568-0020)
Permanent ink markers
Clear tubing (6 inch in length) for gradient mixer outlet (1/16 in diameter)
Wipes (Fisher Scientific, catalog number: 06-666C)
Flat-tip forceps (Fisher Scientific, catalog number: 16-100-116)
Porcelain pestle and mortar (Fisher Scientific, catalog number: S27075)
Ice bucket (Styrofoam box can be used as an alternative)
Basic binder paper clip (medium)
Gloves
Stainless steel spatula with tapered end
Tris (Molecular Grade) (Promega, Fisher Scientific catalog number: PR-H5133)
KCl (Fisher Scientific, catalog number: AC418205000)
MgCl₂ (Fisher Scientific, catalog number: M33-500)
Deoxycholic acid (Fisher Scientific, catalog number: BP349-100)
Polyoxyethylene 10 tridecyl ether (Millipore Sigma, catalog number: P2393-100G)
Cycloheximide (Millipore Sigma, catalog number:01810-G)
Chloramphenicol (Millipore Sigma, catalog number: 220551-25GM)
40 U/mL RNase inhibitor (Promega, catalog number: N2515)
Sucrose, molecular biology grade (Millipore Sigma, catalog number: 573113)
Bromophenol blue (Fisher Scientific, catalog number: AAA1846909)
Dithiothreitol (DTT) (Thermo Fisher Scientific, catalog number: R0861)
Murashige and Skoog (MS) plant basal salt (MP Biomedicals, Fisher Scientific catalog number: ICN2623022)
RNaseZap^TM RNase decontamination solution (ThermoFisher, catalog number: AM9780)
Liquid nitrogen
Aluminum foil

Equipment

Gradient mixer (Buchler Instruments). Catalog number for this specific product is not available as no vendor supports this product anymore. For custom manufacturing of the gradient mixer, see Figure 2.
Tabletop ultracentrifuge (Beckman Coulter, catalog number: A95761 or equivalent)
TLS-55 swinging-bucket rotor with four buckets for up to four gradients (Beckman Coulter, catalog number: 346936)
Gradient station or any equivalent gradient fractionators (Biocomp, catalog number: 153-002)
ISCO UA 5 100 absorbance/fluorescence monitor. Catalog number for this specific product is not available as the vendor is out of market. Alternative product information: BR-188 Density Gradient Fractionation System with Peak Chart Data Acquisition System (Brandel)
Tabletop centrifuge with rotor to hold 1.5 mL tube. If refrigerated centrifuge is not available, then a non-refrigerated centrifuge can be placed in a 4 °C cold room as an alternative
Magnetic plate stirrer
Magnetic stirrer bar (Fisher Scientific, catalog number: 14-513-93). Cut the magnetic stirrer bar into half with the plier and store the halves in a 1.5 mL tube (micro-stirrer bar).
Note: Cleaning of this micro stirrer bar should be done with warm water and stored carefully.
Weighing balance with 0.001 g accuracy
Vortex
Burette stand

Software and Datasets

The BR-188 Density Gradient Fractionation System comes with the Peak Chart Data Acquisition System Software. See note in section E to develop polysome profile without a Peak Chart Data Acquisition System Software.

Procedure

Growing Arabidopsis seedling and tissue storage
This step is a standard procedure in plant biology and various recourses are available that outline the method for germinating Arabidopsis seeds on 1/2× MS plant media. After 12 days of growth, seedlings are harvested into a 1.5 mL tube, flash frozen in liquid nitrogen, and then stored at -80 °C.
Note: It is recommended to use this sample within 1–2 months for best reproducibility of the experiment.

Readiness of gradient maker and general setup (see Figure 1A and Figure 2A for details)
Note: Application of RNaseZap^TM RNase decontamination solution on gloves is recommended before beginning the following procedure.
1. One day prior to the polysome profiling experiment, rinse the gradient maker with diluted dish washing soap in warm tap water for 1–2 min and then wash with warm tap water for few minutes. Micro-stirrer bar needs to be cleaned with just warm water and dried with wipes.
2. Rinse the gradient maker with DEPC-treated water twice and air dry on a few layers of wipes.
3. Arrange the following items on a stable bench: pipettes, tips, burette stand, magnetic stirrer plate, magnetic stirrer bar, 2.2 mL polypropylene centrifuge tubes, 1.5 mL tube stand, trash cans for liquid and solid waste, and stock solutions of 15% and 50% sucrose, CHX, CHL, and DTT.
4. Attach the gradient maker support rod to the burette stand holder and adjust the height of the holder such that the base of the gradient maker is ~0.5–1 inch above the magnetic stirrer plate.
5. Add the micro-magnetic stirrer bar into the chamber connected to the outlet tubing (Well-II) and turn the magnetic stirrer plate ON to confirm rotation of the micro-stirrer bar.
6. Turn the push-pull valve to face towards self, closing the gradient conduit between the mixing chambers (Well-II and Well-I).
7. Clamp a binder clip to the outlet tubing (1 inch away from the gradient maker outlet valve).
8. Attach a 10 µL pipette tip at the end of the outlet tubing using the broad side for the outlet tubing and the tip side for elution.
9. CHL-CHX-DTT mix: Add 15 µL each of CHX and CHL stock and 1.5 µL of DTT stock into the same 1.5 mL tube and mix by vortexing at room temperature for 10–20 s. Store on ice.
  
  Figure 2. Schematics of the gradient mixer. A. Original view of the gradient mixer. The push-pull valve at the bottom of the apparatus is facing towards self, indicating closed connection between the two gradient chambers. B–D. Outline of the gradient mixer in different view angles showing specific measurements. Abbreviations: in = inches, i.d. = inner diameter
Preparation of 2 mL sucrose gradients for pre-run before polysome profiling
1. Add 2.1 µL of CHL-CHX-DTT mix along the side wall of each Well-I and II.
2. Add 1 mL of 15% sucrose solution into Well-I and 1 mL of 50% sucrose solution in Well-II (magnetic stirrer should be rotating in Well-II while 50% sucrose solution is added to Well-II) (see Figure 1A).
3. Remove the binder clamp gently to allow the 50% sucrose solution to flow temporarily approximately 1 cm into the outlet tubing. Quickly clamp back the tubing.
  Note: For a precise control over the flow of the 50% sucrose solution, the tip of the outlet tubing can be carefully inserted into the tip of a 200 µL tip attached to a 20–200 µL pipette. Once the clamp is removed, the pipette is adjusted to allow 50% sucrose solution exactly 1 cm into the outlet tubing. On completion of this step, clamp the tubing again and remove the 200 µL tip, and proceed to the next step.
4. Turn the push-pull valve to face horizontal position, opening the gradient conduit between the mixing chambers (Well-I and Well-II).
5. Air bubbles trapped in the passage between the two wells need to be removed by sealing the top of the Well-I with a gloved finger and then slightly pressing down to increase the pressure.
  Note: This will release air bubbles trapped in the valve passage into the Well-II.
6. While making sure that the outlet is at the same height with the bottom of Well-II, place the tip of the outlet tubing into the bottom of a 2.2 mL ultracentrifuge tube and gently open the clamp. Lower the centrifuge tube slowly and let the solution flow along the walls of the centrifuge tube by gravity. Make sure that the solutions drain evenly from both Well-I and Well-II. When the solution in Well-II is almost over (~200 µL remaining), tilt the gradient maker clockwise until the base of the gradient maker touches the magnetic stirrer plate, while making sure the tip at the end of the outlet tube touches the top of the finished gradient.
  Note: This step will ensure all liquid flows out.
7. Place the finished gradient in a 1.5 mL tube rack and cover it with aluminum foil. Repeat the steps B6–9 and C1–6 for making more gradients.
8. When all the gradients are done, adjust the weight of each gradient tube to 2.800 g by gently adding 15% sucrose solution on top of the finished gradient.
9. For pre-run, place the gradient tube using forceps into a TLS-55 rotor bucket (pre-chilled at 4 °C) and make sure to match the weight with other tubes using 15% sucrose solution.
  Note: It is important to spin all the four buckets of the rotor with equal weights.
10. Spin at 50,000 rpm (213,626.2 × g) for 70 min at 4 °C (deceleration = 0, meaning no brakes).
11. After the run, take the gradient tubes out using forceps and place in a 1.5 mL tube rack. Cover all the gradients with aluminum foil and store at 4 °C on a stable base without any shaking.
  Note: Pre-run gradients are good for up to 24 h.

Cell lysis and sucrose density gradient centrifugation
Note: Two days prior to polysome profiling, wash mortars and pestles with dish soap and rinse with tap water. Use a new set for each sample to prevent cross contamination. Air dry and wrap one mortar with pestle as a pair with aluminum foil and autoclave for 30 min using dry setting. One day before the experiment (day of steps A and B), place the mortar-pestle set at -20 °C (or preferably at -80 °C). (This practice is done to reduce time spent for chilling the mortar-pestle set and save on liquid nitrogen.)
*The term pre-chilled in the following section refers to the use of liquid nitrogen to achieve the coldest temperature.
1. Add liquid nitrogen to the cold mortar-pestle until no boiling of liquid nitrogen is observed. Collect your tissue samples from the -80 °C freezer and keep them floating in a dewar with liquid nitrogen until it is their turn.
2. Add the frozen tissue from the storage tube into the mortar and grind in liquid nitrogen to a fine powder using the pestle. (This step is crucial and usually takes 4–5 min. After every 1 min, add some liquid nitrogen gently into the mortar to keep the ground material cold.) It also helps to have the mortar sit inside a Styrofoam box that is kept cold with liquid nitrogen.
3. Using a pre-chilled spatula, weigh 150 mg of pulverized tissue powder in a pre-chilled 1.5 mL tube.
  Note: During waiting periods, 1.5 mL tube with open cap should be kept in a rack that is placed inside a Styrofoam container with some liquid nitrogen. The cap is left open to allow liquid nitrogen to evaporate. Cover the container to avoid condensation buildup on the top of the 1.5 mL tube.
4. Add 100 µL of freshly prepared lysis buffer (see Recipes) into the 1.5 mL tube with tissue powder and place the tube in a new rack kept at room temperature.
5. After 40–50 s, close the cap of the tube and vortex at highest setting for 1 min at room temperature. Repeat this step twice to achieve complete homogeneity.
6. Spin the tube at 21, 000 × g for 5 min at 4 °C.
7. Transfer 125 µL of the supernatant into a new 1.5 mL tube and repeat centrifugation at 21,000 × g for 5 min at 4 °C.
8. Carefully layer 100 µL of the supernatant from step 7 onto the pre-made 15–50% sucrose gradient (see section C).
  Notes:
  1. Tubes can be numbered with a marker at this stage.
  2. For a blank gradient, layer 100 µL of lysis buffer on a pre-made 15%–50% sucrose gradient.
9. Place the gradient tube using forceps into a TLS-55 rotor bucket (chilled at 4 °C) and make sure to match the weight with other tubes. 15% sucrose solution can be used for this purpose.
  Note: It is very important to use all the four rotor buckets with equal weights.
10. Spin at 50,000 rpm (213,626.2 × g) for 70 min at 4 °C (deceleration = 0, meaning no brakes).
11. Take the gradient tubes out using forceps and place the tubes in a 1.5 mL tube rack (Figure 3A). Cover all the gradients with aluminum foil and place on ice.

Polysome profiling and gradient fractionation
Note: To begin Section E, the gradient should be clamped down in the gradient fractionator and sealed against the top assembly with the outlet tube. (Refer to manufacturer’s manual for details.) The general idea is that the gradient tube gets pierced at the bottom and the heavy push solution is pumped into the bottom of the gradient tube. This way, the gradient is displaced up through the outlet tube at the top into the absorbance recorder and finally out for fractionation.
After each gradient run, the fractionator tubing should be washed with 50 mL of warm water by siphoning it from one end. Dry both the inlet and outlet with wipes and apply vacuum to get rid of any residual water in the fractionator tubing. The baseline absorbance (A_254nm) should return to zero.
1. Run the punch solution through the fractionator tubing at lowest speed (for example, 375 μL/min in the ISCO Teledyne gradient fractionator). Continue pumping the punch solution until it comes out of the piercing needle.
2. Carefully wrap a small piece of parafilm (2 cm wide) around the circumference of a 2.2 mL tube with just water and load the tube into the holder of the gradient fractionator.
  Note: Parafilm is installed for better grip of the tube with the adapter for gradient fractionator.
3. Rotate the collector piston to pierce the gradient tube and start the pump as described in step E1 (Figure 3B).
4. As the water starts to come out of the fractionator tubing, adjust the absorbance (A_254nm) to zero for recording baseline at sensitivity of 0.5 in the ISCO Teledyne gradient fractionator.
5. On completion of the water tube run, take the tube out (see step E6) and load the blank gradient tube (see step D8b) for recording blank reading (Figure 3C).
6. After each gradient run, the fractionator tubing should be washed with 50 mL of warm water by siphoning it from one end. Dry both the inlet and outlet with wipes and apply vacuum to get rid of any residual water in the fractionator tubing. The baseline absorbance (A_254nm) should return to zero.
7. After blank gradient recording is complete, take the tube out and load the first sample gradient tube (Figure 3A). Collect eight 200 µL fractions in individual 1.5 mL tubes. Place tubes on ice immediately after each fraction collection.
  
  Figure 3. Miniature gradient tube setup and polysome profile. A. Gradient tube with sample (Arabidopsis cell extract) after ultracentrifugation. B. Setup of the gradient tube in the ISCO Teledyne gradient fractionator with punch solution inside the tube. C. Example of 15%–50% miniature sucrose gradient. The position of the 40S, 60S, monosome (80S), and the polysome is indicated on the sample gradient profile.

Notes

If a gradient fractionator is not available, individual fractions can be collected by carefully aliquoting 200 µL of the finished gradient from the top using a 20–200 µL pipette. For polysome profile, RNA extracted from these fractions can be quantified using a spectrophotometer and plotted with x-axis as fraction # and y-axis representing absorbance.

Acknowledgments

This project was developed during the postdoctoral tenure of Dr. Ansul Lokdarshi (corresponding author) in the lab of Dr. Albrecht von Arnim (Professor, Dept. of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville). This work was supported by grants from the National Science Foundation (IOS-1456988 and MCB-1546402) and the National Institutes of Health NIH R15 GM129672 to Dr. Albrecht von Arnim. We also thank Ricardo Urquidi Camacho (Graduate Research Assistant, University of Tennessee, Knoxville) for helpful discussions during the optimization of this procedure.

The protocol discussed in this manuscript has been successfully used for polysome profiling (Lokdarshi et al., 2020b and 2020c) and furthermore for RNA and protein analysis from the gradient fractions (Lokdarshi et al., 2020c).

Please see “Figure 8: Ribosome-RNA profile of wild-type and gcn2 mutant under salt stress.”(Lokdarshi et al., 2020b).

Please see “Figure 6. EBP1 associates with cytosolic ribosomes and supports ribosome biogenesis.” (Lokdarshi et al., 2020c).

Competing interests

Authors declare no competing interests.

References

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