2.3.1. Puromycin Incorporation Assay

Treatment with sublethal concentrations of puromycin for a short time prior to harvesting of cells will result in a population of puromycilated polypeptides that are representative of the level of translational output during the given time [51]. A subsequent western blot using an anti-puromycin antibody was normalized to total loaded protein (Figure 3A).

Measuring translational output. (A) Top panel: A western blot of whole lysates from representative samples treated with temperature stress for the indicated time, using an α-puromycin antibody to recognize puromycilated peptides, indicative of translational output. Bottom panel: Total protein for normalization of the α-puromycin signal. (B) A histogram of flow cytometry results following the click chemistry labeling reaction of incorporated HPG. The histogram in green shows basal levels of translational output while the histogram n red demonstrates inhibition of translational output by cycloheximide (CHX) treatment.

Puromycin incorporation assay:

Start 50 mL cultures in baffled flasks at OD₆₀₀ = 0.15–0.20 in the desired media. Incubate cultures, shaking, until mid-logarithmic growth phase is reached, an OD₆₀₀ = 0.55–0.70.

If translation is to be assessed for response to stress or specific compounds, treat cultures appropriately ensuring a no-stress/no-drug control is also analyzed.

10 min prior to your desired time point, pellet cells for 2 min at 4000 RPM, resuspend in a volume of 5 mL of media (smaller volume used to limit the quantity of puromycin used) with a final concentration of 150 μg/mL.

Allow puromycin to incorporate for 10 min.

Pellet cells by centrifuging for 2 min at 4000 RPM and flash freeze cultures in liquid nitrogen.

Cell lysis:

Thaw pellets on ice and resuspend pellet in 30 μL lysis buffer.

HEPES pH 7.4: 15 mM

KCl: 10 mM

MgCl₂: 5 mM

Halt Protease Inhibitor (ThermoFisher): 10 μL/mL

In an Eppendorf Safe-Lock tube, layer 0.5 mL of 0.5 mm glass disruption beads (RPI). Add resuspended pellet to the top of the beads, and layer with another 0.5 mL of beads.

Lyse in Bullet Blender Tissue Homogenizer, chilled with dry ice, for 5 min on speed 12.

Add an additional 50 μL of lysis buffer to lysate and beads, and vortex to mix.

Remove lysate from beads and transfer to a new microfuge tube, and centrifuge at 4 °C for 10 min at 15,000 RCF to clear lysate.

Transfer supernatant to new microfuge tube.

Quantify protein using a Qubit fluorometer protein assay (Invitrogen) or other suitable quantification method.

Western blotting:

To 25 μg of protein, add an equal amount of 2× Laemmli buffer and boil samples at 95 °C for 5 min.

Load samples onto a Bio-Rad Mini-PROTEAN TGX Stain-free gel, 4–15%.

Run the gel for 5 min at 50 V.

Increase the voltage to 120 V and run until the dye front reaches the bottom of the gel.

Remove gel and image total protein using a Bio-Rad GelDoc.

Transfer using Bio-Rad Trans-Blot Turbo as per manufacturer’s instructions.

Block for 5 min in Bio-Rad EveryBlot blocking buffer.

After 5 min of blocking, add α-puromycin antibody (catalog no. MABE343; Millipore) at a 1:1000 dilution.

Incubate overnight at 4 °C.

Wash the blot with Tris-buffered saline containing 0.5% Tween 20 (TBST) for 5 min (repeat ×3).

Incubate with HRP-conjugated α-mouse secondary antibody (catalog no. 7074S; Cell Signaling Technologies) at a 1:10,000 dilution.

Wash the blot with TBST for 5 min (repeat ×3).

Apply chemiluminescent substrate to the blot and image using Bio-Rad ChemiDoc.

Quantify total signal for each lane and normalize to total protein for each respective lane.

This method for assessing translational output does not come without drawbacks, chief among which is that upon incorporation of puromycin into the nascent polypeptide chain, the polypeptide is released prematurely into the cytosol, resulting in a detrimental accumulation of protein products. Additionally, this technique is particularly sensitive to the final concentration of puromycin used and the time allowed for incorporation, so great care needs to be taken to achieve accurate results. Typical concentrations of puromycin used in these assays range from 1–10 μg in mammalian cells and S. cerevisiae. However, application of this technique to C. neoformans required an increased puromycin concentration. It is unclear if this is due to decreased cell permeability to the drug, or if there is some alternative metabolism. We have previously shown that our optimized protocol demonstrated reduction in puromycin incorporation following treatment with hydrogen peroxide, supportive of strong translational repression that was also seen by polysome profiling [10].