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Method Article

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

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DOI:

10.3791/57600

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April 8th, 2018

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 2Department of Biological Sciences, Texas Tech University, 3CISER (Center for the Integration of STEM Education & Research), Texas Tech University

In This Article

Summary

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Abstract

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Introduction

Regulation of gene expression in cells is controlled by transcriptional, posttranscriptional and posttranslational mechanisms. Advances in deep RNA sequencing allow the study of steady-state mRNA levels on a genome-wide scale at an unprecedented level. However, recent findings revealed that steady-state mRNA level does not always correlate with protein production1,2. The fate of an individual transcript is very complex and depends on many factors like internal/external stimuli, stress, etc. Regulation of gene expression during protein synthesis provides another layer of expression control necessary for a rapid response in changing conditions. Polysome (or "polyribosome") profiling, the separation and visualization of actively translating ribosomes, is a powerful method to study the regulation of protein synthesis. Although, its first experimental applications appeared in the 1960s3, polysome profiling is currently one of the most important techniques in protein translation studies4. Single mRNAs can be translated by more than one ribosome leading to the formation of a polysome. Transcripts can be stalled on ribosomes with cycloheximide5 and mRNAs containing different numbers of polysomes can be separated in the process of polysome fractionation by sucrose gradient ultracentrifugation6,7,8,9. RNA analysis of polysomal fractions then allows measurement of changes in the translational states of individual mRNAs on genome-wide scale and during different physiological conditions4,7,10. The method has been also used to reveal the roles of 5'UTR and 3'UTR sequences in control of mRNA translatability11, examine the role of miRNAs in translational repression12, uncover defects in ribosome biogenesis13, and understand the role of ribosome-associated proteins with human diseases14,15. During the last decade, a growing role for regulation of gene expression during translation has emerged that illustrates its importance in human diseases. The evidence for translational control in cancer, metabolic and neurodegenerative diseases is overwhelming15,16,17,18. For example, dysregulation of eIF4E-dependent translational control contributes to autism related deficits15 and FMRP is involved in stalling of ribosomes on mRNAs linked to autism14. Thus, polysomal profiling is a very important tool to study defects in translational regulation in multiple human diseases.

Protein analysis of polysomal fractions under different physiological conditions dissects the function of factors associated with ribosomes during translation. The polysome profiling technique has been used in many species including yeast, mammalian cells, plants, and protozoa10,19,20,21. Protozoan parasites like Trypanosoma and Leishmania exhibit limited transcriptional control of gene expression. Their genomes are organized into polycistronic gene clusters that lack promoter-regulated transcription22. Instead, developmental gene expression is predominantly controlled at the level of protein translation and mRNA stability in trypanosomatid species23,24. Therefore, understanding of translational control in the absence of transcriptional regulation is particularly important for these organisms. Polysomal profiling is a powerful tool to study posttranscriptional regulation of gene expression in Leishmania25,26,27,28.

The recent progress in detection of individual mRNAs levels by real time quantitative PCR (RT-qPCR) and full transcriptome by next-generation sequencing, as well as proteomics technologies, brings resolution and advantages of polysomal profiling to a new level. The use of these methods can be further extended by analysis of individual polysomal fractions by deep RNA sequencing combined with proteomic analysis to monitor the translational status of cells on a genome-wide scale. This allows the identification of new molecular players regulating translation under different physiological and pathological conditions. Here, we present a universal polysome profiling protocol used on three different models: the parasite Leishmania major, cultured human cells, and animal tissues. We present advice on the preparation of cell lysates from different organisms, optimization of gradient conditions, choice of RNase inhibitors and application of RT-qPCR, Western blot and RNA electrophoresis to analyze polysome fractions in this study.

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Protocol

All animal treatments and handling of tissues obtained in the study were performed according to protocols approved by the Institutional Animal Care and Use Committee at the Texas Tech University Health Science Center in accordance with the National Institutes of Health animal welfare guidelines, protocol number 96005. Please sacrifice vertebrate animals and prepare tissues according to the guidelines from the Institutional Animal Care and Use Committee. If lacking such a committee, please refer to the National Institutes of Health animal welfare guidelines. Adult (>60 day old) C57BL/6 mice were used. All animals and tissues were obtained according to protocols approved by the Institutional Animal Care and Use Committee at the Texas Tech University Health Sciences Center in accordance with the National Institutes of Health animal welfare guidelines. For euthanasia, a single mouse was placed in a small chamber, and the air was displaced gradually with about 30% carbon dioxide to anesthetize and minimize the distress of the animal. Following cessation of breathing, we used cervical dislocation to confirm the death of the animal before harvesting tissues.

Caution: All work with live Leishmania and cultured human cells was done in biosafety cabinet in BSL-2 certified laboratory.

1. Preparation of Cytoplasmic Lysates from Leishmania Major , Cultured Human Cells, and Mouse Tissues

NOTE: There are several differences in the lysate preparations from the different source materials. Other steps including sucrose gradient preparation and polysomal fractionation are identical and do not depend on sample source.

  1. Leishmania major cytoplasmic lysate preparation
    1. Inoculate Leishmania major (FV1 strain) cells in 30 mL of 1x M199 medium29 containing 10% Fetal Bovine Serum (FBS) and penicillin/streptomycin mixture (100 units and 100 μg/mL correspondingly) at density of 1x105 cells/mL.
      NOTE: All steps involving Leishmania major cells must be conducted in a biosafety cabinet.
    2. Place cells in the incubator and grow them at 27 °C until the logarithmic phase (mid log corresponds to 5x106 cells/mL). It usually takes about two days to grow.
    3. Add cycloheximide to Leishmania major culture to a final concentration of 100 μg/mL to arrest the ribosomes on translated mRNAs. Place cells back in the incubator for 10 min at 27 °C.
    4. After cycloheximide treatment is completed, transfer cells to a 50 mL conical tube and spin them at 1,800 x g and 4 °C for 8 min. Discard supernatant.
    5. Wash cells with 30 mL of Dulbecco's phosphate buffered saline (DPBS). Centrifuge at 1,800 x g and 4 °C for 8 min.
    6. Discard the supernatant. Resuspend cells in 1 mL of DPBS.
    7. Take an aliquot of cells and mix it with 3.5% formaldehyde solution.
    8. Count cells by hemocytometer and determine their concentration. Transfer the desired number of cells into microfuge tube. Lysate prepared from 0.5x108-2x108 cells/mL is sufficient for one sucrose gradient loading.
    9. Spin the cells at 1,800 x g and 4 °C for 8 min. Discard the supernatant.
    10. Resuspend the cell pellet on ice in 1 mL of lysis buffer containing protease inhibitors and RNase inhibitor (20 mM HEPES-KOH, pH 7.4, 100 mM KCl, 10 mM MgCl2, 2 mM DTT, 1% NP-40, 1x protease inhibitor cocktail (EDTA-free), 200 units/mL RNase inhibitor).
    11. Pass the lysate through a 23-gauge needle three times. The lysate should become transparent after passage through the needle.
    12. Centrifuge at 11,200 x g and 4 °C for 10 min to clarify lysate. Transfer the clarified lysate to a fresh tube and keep it on ice until sucrose gradient ultracentrifugation.
    13. Collect 400-500 μL of the lysate as input (to analyze later), freeze it right away in liquid nitrogen for future protein analysis or add RNA purification reagent before freezing for RNA analysis.
  2. Cytoplasmic lysate preparation from cultured human HeLa cells
    1. Split HeLa cells and seed them into 20 mL of the DMEM medium containing 10% FBS and penicillin/streptomycin mixture (100 units and 100 μg/mL correspondingly) with cell count 2x105 cells/mL in a 15 cm plate.
    2. Grow HeLa cells at 37 °C, 5% CO2 for 20-24 h. Perform plasmid DNA transfection according to manufacturer's protocols.
    3. Propagate cells for 24 h after transfection at 37 °C, 5% CO2.
    4. Add cycloheximide to grown HeLa cells to the final concentration of 100 μg/mL to arrest the ribosomes on translated mRNAs and incubate cells for 10 min at 37 °C, 5% CO2. Aspirate medium. Wash the cells twice with cold DPBS on ice.
    5. Add 500 μL of lysis buffer (20 mM HEPES-KOH pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.5% NP-40, 1x protease inhibitor cocktail (EDTA-free), 200 units/mL of RNase inhibitor or 1 mg/mL heparin) to the plate and scrape the cells on ice.
    6. Transfer the lysed cells to the microfuge tube. Adjust the concentration of NP-40 to 0.5% and MgCl2 to 5 mM according to the increased volume of the sample.
    7. Pass the lysate through a 23-gauge needle 3-6 times.
    8. Spin at 11,200 x g and 4 °C for 8 min to clarify lysate. After centrifugation, transfer supernatant to a new tube. Use a spectrophotometer to evaluate cell lysis efficiency and to determine sample amount for the loading on the gradient. Add 10 μL of sample to 0.5 mL of 0.1% Sodium Dodecyl Sulfate (SDS). Blank against 0.1% SDS. Measure absorbance at 260 nm. Expected absorbance value is around 15-20 units/mL.
    9. Dilute all samples with lysis buffer to the same absorbance value before sucrose gradient centrifugation. Keep samples on ice until sucrose gradient centrifugation.
  3. Cytoplasmic lysate preparation from mouse testis
    1. Dissect the mouse testis. Make a small incision in the tunica albuginea and collect the seminiferous tubules of the testes and transfer them in a 15 mL conical tube containing 5 mL of DPBS supplemented with 0.1 mM phenylmethylsulfonyl fluoride (PMSF).
    2. Mix the tissue vigorously by inverting several times. Allow the tissue to settle at unit gravity on ice for 5 min.
    3. Remove and discard the cloudy buffer containing connective cells and tissue fragments. Repeat the procedure 2-3 more times. The remaining white pellet is enriched for seminiferous tubules and germ cells.
    4. Transfer the seminiferous tubule pellet to a 2 mL microcentrifuge tube and spin at 500 x g for 1 min. Discard the supernatant.
    5. Add 500 µL of lysis buffer (20 mM Tris-HCl, pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.5% NP-40, 1x protease inhibitor cocktail (EDTA-free), 1 mg/mL heparin or 200 units/mL of RNase inhibitor) to the tubules. Use a pipette to triturate the tissue.
    6. Transfer the suspension to a small (0.5-1.0 mL) Dounce homogenizer. Disrupt the tissue with seven to eight strokes of the glass pestle.
    7. Transfer the lysate to a 1.5 mL microcentrifuge tube.
    8. Centrifuge the sample at 12,000 x g and 4 °C for 8 min to clear the lysate. Transfer the supernatant to a new tube and store on ice until loading on the sucrose gradient.
    9. Collect 50 μL of the lysate as input sample, freeze it right away at -80 °C for future protein analysis; or add RNA purification reagent before freezing for RNA analysis.

2. Sucrose Gradient Preparation and Ultracentrifugation

  1. Prepare two sucrose gradient solutions (20 mM HEPES-KOH, pH 7.4, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 1x protease inhibitor cocktail), containing either 10% sucrose or 50% sucrose. (Tris-HCl, pH 7.4, can be used instead of HEPES). Add 200 units/mL RNase inhibitor or 1 mg/mL heparin according to the experimental design. Place an ultracentrifuge tube for SW 41 rotor into the marker block and draw the line along the upper level of the block. Transfer the tube into a stable rack.
  2. Take a 10-mL syringe with the layering device attached and fill the syringe with 10% sucrose solution (prepared as above). Gently release it at the bottom of the ultracentrifuge tube until it reaches the mark on the tube.
  3. Fill another syringe with 50% sucrose solution and carefully insert its layering device through the 10% sucrose layer to the bottom of the tube. Gently release sucrose solution starting from the bottom until it reaches the mark on the tube. Seal the tube with the provided cap.
  4. To prepare the sucrose gradient, turn the gradient maker device ON. Level the plate using the UP or DOWN buttons and press DONE. Leveling is important for linearity of the gradient.
  5. After leveling the plate press GRAD to open the gradient menu. Go to LIST on the gradient menu and select the SW 41 Ti rotor. Then choose the desired sucrose gradient from the list of the menu using UP and DOWN buttons. Press USE.
  6. Place the gradient tube holder on the gradient maker plate. Transfer the tube into the holder. Up to 6 gradients can be prepared at the same time. Press RUN. The gradient maker rotates the tubes at the programmed speed and angles forming a linear gradient. It will take only a few minutes to prepare the gradient.
  7. When process is completed, place the tubes in a rack. Take the caps off. Remove the same volume as the sample volume from the top of the ultracentrifuge tubes.
  8. Carefully load 400-500 μL of lysate containing 15-20 A260 units of polysomes on the top. Place the tubes in the rotor buckets and balance them.
  9. Centrifuge at 260,000 x g and 4 °C for 2 h using SW 41 rotor.

3. Polysome Fractionation and Sample Collection

NOTE: While lysate preparations have some differences depending on the source, gradient preparation and polysome fractionation protocols are the same for all types of lysates.

  1. After completion of the ultracentrifugation, place the rotor buckets with the tubes on ice. Turn fraction collector and gradient fractionator ON. Click SCAN on the fractionator menu. Put a rack with 24 collection tubes into the fraction collector.
  2. Fill up a rinse reservoir on the side of the fractionator with deionized water. Press the RINSE key for 10 s to rinse the pump on fractionator. Attach a rinse adaptor with the syringe filled with water to the piston for the calibration.
  3. Open the fractionator software on the computer. Press CALIBRATE. Use the DEFAULT settings and press OK. Be ready to inject water from syringe.
  4. Press OK to do calibration. Immediately start injecting water for the next 5 s. During this time, water will flow through the UV detector flow cell and the instrument will be calibrated. The sign ZERO CALIBRATION COMPLETED will appear. The instrument is ready for fractionation.
  5. Remove the rinse adaptor with the syringe. Attach a tip to the piston of the fractionator.
  6. Open the brass air valve and press AIR key for 10 s to dry tubing and flow cell. Close the air valve.
  7. Gently remove the gradient tube from the rotor bucket and place it in the rack. Apply the tube holder cap to the top of the tube and carefully move the tube into the tube holder and lock it in position.
  8. Place the holder under the piston of the fractionator. Often, polysomal bands can be seen by eye. Introduce the desired settings for fraction numbers and volume (24 fractions at 500 μL/fraction are usually sufficient). Name the file appropriately. Press OK, and then GO TO GRAPH button. In the next window, press START SCAN. Settings will appear, press OK. The collector will move from the gutter to the first fraction and the piston will move into the tube. When the piston reaches the top of the gradient it will slow to the selected speed and the fractions will be collected. When completed, the piston moves out of the gradient tube.
  9. Open the brass air valve and press AIR key on the fractionator to retrieve the last fraction.
  10. Move the tubes from the rack on ice.
  11. Add 2 volumes of RNA purification reagent to each fraction and flash freeze in liquid nitrogen until RNA purification. Alternatively, if protein needs to be analyzed, add trichloroacetic acid to the final concentration of 10% to concentrate them for Western blotting (see Section 8).

4. Preparation of Synthetic RNA In Vitro for Normalization of mRNAs Levels During RT-qPCR Data Analysis

NOTE: The E. coli OmpA mRNA is used in this protocol for normalization. Any other RNA that does not have extensive identity with the mRNAs of the studied organism (mammalian or Leishmania) can be used.

  1. Prepare the OmpA DNA fragment containing SP6 promoter sequence by a standard PCR reaction from a plasmid containing OmpA gene30.
  2. Prepare 100 µL of the mixture: 80 mM HEPES-KOH, pH 7.5, 16 mM MgCl2, 2 mM Spermidine, 10 mM DTT, 3 mM ATP, 3 mM CTP, 3 mM UTP, 3 mM GTP, 0.5 U/μL RNase inhibitor, 1 μg of OmpA PCR DNA, 3 μL SP6 RNA polymerase, 0.005 U/μL pyrophosphatase.
  3. Incubate at 40 °C for 2 h.
  4. Purify RNA by a RNA purification kit.
  5. Measure concentration by spectrophotometer and examine by agarose gel electrophoresis.

5. RNA Isolation from Gradient Fractions and cDNA Preparation

NOTE: Proceed directly with this protocol for RNA purification if an RNase inhibitor was used as a ribonuclease inhibitor. However, when used as a ribonuclease inhibitor, heparin will inhibit reverse transcriptase used in cDNA preparation. Therefore, additional purification of RNA will be needed if heparin was used in the lysis buffer and gradient. See Section 6 to prepare RNA for cDNA synthesis if heparin was used.

  1. Thaw the samples containing RNA purification reagent, add 20 ng of synthetic RNA as internal control for normalization of RT-qPCR results. Proceed with RNA preparation according to the manufacturer's protocol except one modification. Add 1 µL of RNA grade glycogen (20 µg) prior isopropanol precipitation. Dissolve RNA pellets in 20-25 µL of RNase-free water.
    NOTE: Glycogen serves as a carrier and helps to avoid losses and visualize RNA pellet during purification. OmpA mRNA is used for further normalization in RT-qPCR reactions.
  2. Measure RNA concentration using spectrophotometer to ensure adequate yield. Combine equal volumes of RNA fractions containing 40S, 60S and monosomes as prepolysomes. Fractions containing 2-4 ribosomes combine as light polysomes and fractions with 5-8 ribosomes combine as heavy polysomes.
  3. Use 5-10 µL of RNA from combined fractions to prepare cDNAs using a kit and following manufacturer's recommendations.
  4. Add 80 µL of nuclease free water to 20 µL of cDNA. Freeze the cDNA samples at -20 °C.

6. RNA Purification from Heparin Contamination

NOTE: Heparin inhibits nucleic acid processing enzymes such as reverse transcriptase. Therefore, use this additional purification protocol when heparin is used in the lysis buffer and/or in the gradient.

  1. Add LiCl to a 1 M final concentration to the purified RNA samples.
  2. Mix the samples and incubate on ice for 1 h.
  3. Spin the samples at 16,000 x g and 4 °C for 15 min.
  4. Remove the supernatant as complete as possible using a pipette.
  5. Air-dry the pellets for about 5 min.
  6. Re-suspend the pellets in the initial volume of RNase-free water.
  7. Perform spectrophotometric measurement at 260 nm to determine the concentration of the RNA. Usually, the loss of the sample is minimal.

7. RT-qPCR and Data Analysis of mRNA Distribution

  1. Combine 10.2 μL of water, 20 μL of SYBR Green, 4.8 μL of gene specific primers (2.5 μM each set), 5 μL of cDNA, mix well and load 10 μL per well in triplicates into 384-well plate.
  2. Cover plate with adhesive film tightly and centrifuge plate at 1,800 x g for 5 min.
  3. Using a Real-Time PCR instrument set up qPCR reaction under conditions shown in the Table 1.
  4. Using the cycle threshold (CT) values and the comparative CT (ΔΔCT) method31 calculate the percentage (%) of mRNA distribution in prepolysomes, light, and heavy polysomes as described32 with one modification. Use synthetic RNA (OmpA here) for data normalization in RT-qPCR data analysis. The synthetic RNA provides a normalization control that allows to calculate relative mRNAs levels and compare them in different fractions of a gradient.

8. Analysis of Proteins in Polysomal Fractions by Western Blotting

  1. From a 100% (w/v) stock, add trichloroacetic acid (TCA) to the selected fractions (500 μL) to a final concentration of 10%, keep on ice for at least 15 min, centrifuge in a microfuge for 5 min, discard supernatant, wash twice with ice-cold acetone and dissolve in 25 μL of SDS-PAGE sample loading buffer for electrophoresis.
  2. Load on the SDS-PAGE and conduct standard electrophoresis with following transfer to the PVDF membrane. Proceed to Western blotting33.

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Results

In this study, we describe the application of the polysomal profiling technique to three different sources: parasitic Leishmania major, cultured human cells, and mouse testis. Leishmania cells freely grow in the liquid media in suspension, cultured human cells grow in the adherent monolayer on plates, and the mouse testis represents a tissue sample. The method can be easily adjusted to other types of freely grown cells in suspension, different types of tissues, or from a...

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Discussion

Polysome fractionation by sucrose gradient combined with RNA and protein analysis of fractions is a powerful method to analyze translational status of individual mRNAs or the whole translatome as well as roles of protein factors regulating translational machinery during normal physiological or disease state. Polysomal profiling is an especially suitable technique to study translational regulation in organisms such as trypanosomatids including Leishmania where transcriptional control is largely absent and gene ex...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors thank Ching Lee for help with audio recording.The research was supported by the Start-up funds from Texas Tech University Health Sciences Center and by the Center of Excellence for Translational Neuroscience and Therapeutics (CTNT) grant PN-CTNT 2017-05 AKHRJDHW to A.L.K.; in part by NIH grant R01AI099380 to K.Z. James C. Huffman and Kristen R. Baca were CISER (Center for the Integration of STEM Education & Research) scholars and were supported by the program.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Instruments:
Gradient masterBiocomp Instruments Inc.108
Piston Gradient FractionatorBiocomp Instruments Inc.152
Fraction collectorGilson, Inc.FC203B
NanoDrop OneThermo ScientificNanoDrop One
Nikon inverted microscopeNikonECLIPSE Ts2-FL/Ts2
2720 Thermal CyclerApplied Biosystems by Life Technologies4359659
CO2 incubatorPanasonic Healthcare Co.MCO-170A1CUV
HERATHERM incubatorThermo Scientific51028063
Biological Safety Cabinet, class II, type A2NuAire Inc.NU-543-400
Revco freezerRevco TechnologiesULT1386-5-D35
Beckman L8-M UltracentifugeBeckman CoulterL8M-70
CentrifugeEppendorf5810R
CentrifugeEppendorf5424
Ultracentrifuge Rotor SW41Beckman Coulter331362
Swing-bucket rotorEppendorfA-4-62
Fixed angle rotorEppendorfF-45-30-11
Quant Studio 12K Flex Real-Time PCR machine 285880228Applied Biosystems by life technologies4470661
TC20 Automated cell counterBio-Rad145-0102
HemacytometerHausser Scientific02-671-51B
Software 
Triax software Biocomp Instruments Inc.
Materials:
Counting slides, dual chamber for cell counterBio-Rad145-0011
1.5 mL microcentrifuge tubeUSA Scientific1615-5500
Open-top polyclear centrifuge tubes, (14 mm x 89 mm)Seton Scientific7030
Syringe, 5 mLBD309646
BD Syringe 3 mL23 Gauge 1 Inch NeedleBD10020439
Nunclon Delta Surface plate, 14 cmThermo Scientific168381
Nunclon Delta Surface plate, 9 cmThermo Scientific172931
Nalgene rapid-flow 90mm filter unit, 500 mL, 0.2 aPESThermo Scientific569-0020
BioLite 75 cm3 flasksThermo Scientific130193
Nunc 50 mL conical centrifuge tubesThermo Scientific339653
Chemicals:
Trizol LSAmbion by Life Technologies10296028
HEPESFisher ScientificBP310-500
Trizma baseSigmaT1378-5KG
Dulbecco's Modified Eagle's Medium-high glucose (DMEM)SigmaD6429-500ML
Fetal Bovine Serum (FBS)SigmaF0926-50ML
Penicillin-Streptomycin (P/S)SigmaP0781-100ML
Lipofectamine 2000Invitrogen11668-019
Dulbecco's phosphate buffered saline (DPBS)SigmaD8537-500ML
Magnesium chloride hexahydrate (MgCl2x6H2O)Acros OrganicsAC413415000
Potassium Chloride (KCl)SigmaP9541-500G
Nonidet P 40 (NP-40)Fluka (Sigma-Aldrich)74385
Recombinant Rnasin Ribonuclease InhibitorPromegaN2511
Heparin sodium saltSigmaH3993-1MU
cOmplete Mini EDTA-free protease inhibitorsRoche Diagnostics11836170001
GlycogenThermo ScientificR0551
WaterSigmaW4502-1L
CycloheximideSigmaC7698-1G
ChloroformFisher Scientific194002
Dithiotreitol (DTT)Fisher ScientificBP172-5
Ethidium BromideFisher ScientificBP-1302-10
Ethylenediaminetetraacetic acid disodium dehydrate (EDTA)Fisher ScientificS316-212
OptimemLife Technologies22600050
Puromycin dihydrochlorideSigmaP8833-100MG
SucroseFisher ScientificS5-3KG
Trypsin-EDTA solutionSigmaT4049-100ML
Hgh Capacity cDNA Reverse Transcriptase KitApplied Biosystems by life technologies4368814
Power SYBR Green PCR Master MixApplied Biosystems by life technologies4367659
HClFisher ScientificA144SI-212
IsopropanolFisher ScientificBP26324
Potassium Hydroxide (KOH)Sigma221473-500G
Anti-RPL11 antibodyAbcamab79352
Ribosomal protein S6 (C-8) antibodySanta Cruz Biotechnology Inc.sc-74459
1xM199SigmaM0393-10X1L
Lithium clorideSigmaL-9650
Dimethyl sulfoxide (DMSO)Fisher ScientificD128-500
Gel Loading Buffer IIThermo ScientificAM8546G
UltraPure AgaroseThermo Scientific16500-100
Trichloracetic acid (TCA)Fisher ScientificA322-100
SuperSignal West Pico PLUS chemiluminescent substrateThermo Scientific34580
FormaldehydeFisher ScientificBP531-500
Sodium Dodecyl Sulfate (SDS)SigmaL5750-1KG
Phenylmethylsulfonyl fluoride (PMSF)SigmaP7626-5G
RNeasy Mini kitQiagen74104
Adenosine 5′-triphosphate disodium salt hydrate (ATP)SigmaA1852-1VL
Cytosine 5'-triphosphate disodium salt hydrate (CTP)SigmaC1506-250MG
Uridine 5'-triphosphate trisodium salt hydrate (UTP)SigmaU6625-100MG
Guanosine 5'-triphosphate sodium salt hydrate (GTP)SigmaG8877-250MG
SP6 RNA PolymeraseNEBM0207S
PyrophoshataseSigmaI1643-500UN
SpermidineSigmaS0266-1G

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Sucrose GradientUltracentrifugationLysate PreparationFraction CollectionRNA AnalysisWestern BlotRibosome AssociationTranslation RegulationModel Organisms
Polysome Profiling in Leishmania, Human Cells and Mouse Testis
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