Polysome and Puromycin immunoprecipitation

Materials:

1. RiboTag expressing cells (HA tagged eukaryotic ribosomal protein L22) 

2. Puromycin (catalog #P7255)
   

3. EDTA-free protease inhibitor cocktail (Roche, catalog #5056489001)
   

4. SUPERaseIN (Invitrogen, catalog #AM2696)
   

5. Dynabeads Protein G (Invitrogen, catalog #10004D)
   

6. Anti-Puromycin, mouse monoclonal 12D10 (MilliporeSigma, catalog #MABE343, RRID:AB_2566826)
   

7. Anti-HA, rabbit polyclonal (Abcam, catalog #ab9110, RRID:AB_307019)

Buffers and solutions:

1. Puromycin diluted to 200µM in the appropriate cell culture medium (2x final concentration). Pre-warm to 37C prior to starting the experiment.

2. Polysome Buffer (150 mM KCl, 10 mM MgCl2, 5 mM HEPES pH 7.4, 1% Igepal CA-630) supplemented with protease inhibitors (1 tablet / 50mL) and SUPERaseIN (0.2 U/µL). Pre-chill on ice for at least 20 minutes prior to starting the experiment.
   

3. Laemmli Sample Buffer (BioRad, catalog #161–0747)
   

4. RLT buffer and purified using RNEasy MinElute columns (Qiagen, catalog #74204).

Procedures:

Cell treatment with puromcyin

Note that this protocol was designed for rapid, high-efficiency puromycin labeling of nascent polypeptide chains undergoing active translation during a short time window (5 minutes). Accordingly, a high puromycin concentration is employed (100 µM), which results in complete release of all nascent polypeptide chains in less than 5 minutes. This puromycin concentration is likely high enough to prevent new synthesis of medium- to full-length nascent polypeptide chains for further rounds of labeling. In other words, after the initial set of nascent polypeptide chains are labeled and released from ribosomes, subsequent polypeptides formed via new translation events will likely be highly truncated by rapid further reaction with puromycin. If longer term labeling of nascent polypeptide chains is desired, such as a 30-minute incubation period where ribosomes continually synthesize and release medium- to full-length polypeptides, a much lower concentration of puromycin must be employed. It has been reported that at sufficiently low puromycin concentrations, puromycin preferentially incorporates towards the C-terminus of actively translating proteins (Miyamoto-Sato et al., 2000). Users should optimize concentrations and incubation times for their experimental goals. 

1. Remove half the media volume from the cell culture. Replace with an equal volume of pre-warmed media containing 200µM puromycin (final concentration will be 100µM). Return cells to incubator and incubate for 5 minutes. 

2. Remove cells from the incubator and place on ice. Gently aspirate the media and add an appropriate volume of ice cold polysome buffer supplemented with protease and RNAse inhibitors (~1 mL for each well of a 6 well-plate). Scrape cells and transfer to a pre-chilled 1.5 mL eppendorf tube. Vigorously pipette the cells with a P1000 pipette >20 times in order to facilitate lysis.

3. Incubate the polysome lysates at 4C on a spinning rotor for at least 15 minutes to complete lysis and release of cytoplasmic polysomes.

4. Centrifuge lysates for 15 minutes at 16,000xg at 4C. Transfer supernatant to a fresh eppendorf tube.

5. Split each clarified lysate for immunoprecipitation of labeled nascent polypeptide chains (anti-puromycin) or of polysomes (anti-HA). Lysates are stable on ice.

Immunoprecipitation

1. Remove Dynabeads Protein G from 4C storage and aliquot an appropriate volume into a fresh eppendorf tube. The amount of Dynabeads Protein G needed will depend on the amount of cell lysate and antibodies used for immunoprecipitation. Wash Dynabeds Protein G at least two times in >1 mL of polysome buffer. Resuspend in 50-100 µL of polysome buffer at room temperature.
   

2. Add an excess of primary antibodies (Rabbit anti-HA or Mouse anti-Puromycin) to the Dynabeads Protein G in 50-100 µL of polysome buffer and incubate for 20 minutes at room temperature. Refer to the manufacturer's instructions for IgG binding capacity of Dynabeads Protein G.
   

3. Remove unbound primary anitbodies by washing Dynabeads Protein G three times in >1 mL polysome buffer. Resuspend in 50-100 µL of polysome buffer and place on ice. The Protein G on the surface of the Dynabeads is now saturated with anti-HA or anti-puromycin antibody and ready for immunoprecipitation.

4. Add antibody-bound beads to clarified polysome lysates. Mix thoroughly by inversion and place eppendorf tubes on a spinning rotor at 4C. Incubate for one hour in order to capture polysomes (anti-HA) or labeled nascent polypeptides (anti-puromycin). Antigen capture can also occur overnight.

5. Place Dynabeads Protein G onto a magnetic rack and remove the supernatant. This supernatant contains the unbound material, and can be subsequently analyzed in order to optimize the % of the target protein captured in the IP. Resuspend the beads in 1 mL ice-cold polysome buffer supplemented with protease and RNAse inhibitors (as above for cell lysis).

6. Return the beads to the spinning rotor at 4C and incubate for >15 minutes. Repeat for a total of four washes over one hour.

7. After the final wash, beads can be eluted for RNA or protein analysis. For RNA analysis, resuspend the beads in 350 µL of RLT buffer and purify using RNEasy MinElute columns as per the manufacturer's instructions. 

8. For protein analysis, the elution strategy will depend on the downstream experimental goals. For western blot analysis, we elute protein from the beads by boiling for 5 min at 95°C in the desired volume of 1x Laemmli Sample Buffer. Eluted proteins can be stored at −80°C until western blotting. Note that elution in 1x Laemmli Sample Buffer is likely to be incompatible with many downstream proteomic analyses.

References:

1. Miyamoto-Sato E, Nemoto N, Kobayashi K, Yanagawa H. Specific bonding of puromycin to full-length protein at the C-terminus. Nucleic Acids Res. 2000;28:1176–1182.

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