Example Experiment: Characterizing ERK Activation in Response to Phorbol 12-Myristate 13-Acetate (PMA)

Background

Accurate quantification of protein expression and/or post-translational modifications is important for advancing both basic and translational research. Given the flexibility, reproducibility, and higher throughput of the In‑Cell Western™ Assay, it offers a convenient alternative to Western blotting and is a powerful platform for meaningful in situ analyses. The In-Cell Western microplate format can be used to analyze:

  • Protein phosphorylation and signaling (1 - 3)
  • Off-target effects of drugs on signaling pathways (4)
  • Timing and kinetics of signaling events (5, 6)
  • Quantification of viral load (7 - 11)
  • Genotoxicity assays (12, 13)
  • Cell proliferation and apoptosis assays (14)
  • Bacterial-induced epithelial signaling (15)
  • Glycoprotein analysis (16, 17)
  • Library screening (18 - 20)
  • Screening of monoclonal antibody clones (21)

Introduction

The protein kinase C (PKC) pathways regulate numerous processes such as, cellular proliferation, gene expression, and the inflammatory response (22). Due to the multifaceted roles of the PKC signaling pathway, it is not surprising that the perturbation of this pathway can promote malignant behavior in cancer cells, such as increased cell survival, proliferation, migration, and the epithelial-mesenchymal transition (23). Therefore, exploring PKC and its corresponding signaling pathways can provide critical knowledge in understanding its role in both normal and pathological processes.

Experimental Design

In the following In-Cell Western example, we present a protocol for monitoring ERK phosphorylation in response to stimulation by phorbol 12-myristate 13-acetate (PMA), a potent nanomolar activator of protein kinase C (PKC) (24). PMA acts by binding to the C1 domain of PKC, thereby triggering a signal transduction cascade that initiates a variety of biochemical and molecular changes, including the phosphorylation and activation of ERK (25, 26). Changes in phospho- and total-ERK protein levels are detected and characterized in 3 suspension cell lines (Jurkat, K-562, and THP-1) treated with a serial dilution of PMA. The information obtained from this experimental protocol could be used to identify appropriate experimental conditions to screen for and characterize compounds that modulate this branch of the PKC signaling pathway.

Required Reagents

LI‑COR Reagents

  • IRDye® 800CW Goat anti-Rabbit Secondary Antibody (LI-COR P/N 925-32211 or 926-32211)

  • IRDye 680RD Goat anti-Mouse Secondary Antibody (LI-COR P/N 925-68070 or 926-68070)

  • Intercept® (PBS) Blocking Buffer

    Odyssey® Blocking Buffer was used in the original experiment and has been discontinued. Intercept Blocking Buffer is now available instead of Odyssey Blocking Buffer.

  • Large Western Incubation Box (LI-COR P/N 929-97301)

Additional Reagents

  • 1X PBS wash buffer

  • Tissue culture reagents (RPMI 1640, fetal bovine serum)

  • Black-sided 96-well or 384-well microplates with clear well bottoms

  • Jurkat cells (ATCC®, P/N TIB-152™)

  • THP-1 monocytes (ATCC, P/N TIB-202™)

  • K-562 lymphocytes (ATCC, P/N CCL-243™)

  • Concentrated Prefer (5X) (Anatech LTD, P/N 411)

  • TO-PRO®-3 (Molecular Probes, P/N T-3605)

  • PMA (phorbol 12-myristate 13-acetate) (Sigma®, P/N P1585)

  • DMSO (dimethyl sulfoxide) (Sigma, P/N D8418)

  • ERK rabbit antibody (Santa Cruz, P/N sc-94)

  • pERK mouse antibody (Cell Signaling Technology, P/N 9106)

Prepare Cells

  1. Allow Jurkat (ATCC, P/N TIB-152) cell growth in a T75 flask using standard tissue culture procedures. Avoid growing cells to density greater than 2 x 106 cells.

  2. Transfer cells in growth media to 50 mL conical tubes and centrifuge at 500 x g for 5 minutes.

  3. Remove media and resuspend cell pellet in 10 mL of serum-free media (pre-warmed to 37 °C). Pipet slowly to maintain cell integrity while disrupting the cell pellet. Transfer resuspended cells into T75 flask and place in an incubator (37 °C and 5% CO2).

    It is the serum withdrawal from the complete media that allows suspension cells to attach to plates (i.e., T75 flask). Gravity will cause cells to form a monolayer over time (10 to 15 minutes). Once a monolayer is formed, the rest of the cells in the serum-free media will remain in suspension and will not attach to the plates once a monolayer of cells are established. Only the cells in suspension in the T75 flask will be used in the following steps.

  4. Allow cells to settle for 30 minutes before taking a 50 µL aliquot of cells for counting using a hemocytometer.

  5. Add appropriate volume of serum-free media so that 1 x 106 cells/mL is achieved (1 plate x 96 wells x 200 µL of cells/well = 20 mL/plate).

  6. Serum-deprive cells by replacing cells suspended in serum-free media back into the incubator for an additional 3.5 hours or overnight.

Treat Cells

  1. Add 2 µL of DMSO for both the background samples (serves as non-specific background fluorescence) and resting cells (serves as basal control) in triplicate wells. Add 2 µL of 1:1 serial dilutions of PMA ranging from 0.005 to 3.2 ng/mL in triplicate wells.

  2. Using a multi-channel pipettor, transfer 200 µL of suspended cells (~200,000 cells) per well into the wells containing 2 µL of DMSO or PMA from step 6.

  3. Allow incubation at 37 °C for 15 minutes.

    Be careful not to disrupt cells during this PMA-induced activation step. During this critical step, cells will sediment to the bottom of the wells by gravity, forming a monolayer. This monolayer can be easily viewed under a light source. The monolayer will appear opaque rather than transparent. Clumping of cells will lead to detachment from plates during incubation and washing steps. Be careful in handling the plate at this stage because the cells will be very loosely attached to the bottom of the wells.

Fix and Permeabilize Cells

Fix Cells

  1. Directly add to the cell suspension 50 µL of concentrated (5X) Prefer (or 25 µL of 37% formaldehyde to the cell suspension; final concentration of 4%) into each well.

    Gently add Prefer into wells using side of the wells to avoid detaching the cells from the well bottom. During fixation, the cell monolayer will attach more firmly to the wells; however, the strength of the attachment is never as strong as that of adherent cells grown on plates. Therefore, a degree of caution is needed during every step of this procedure.

  2. Allow cells to fix for 20 minutes at room temperature with very gentle rotation (set at speed 2 on The Belly Dancer® (Stovall)).

  3. Centrifuge at 1,500 rpm (332 rcf) for 10 minutes.

Permeabilize Cells

Wash 3 times with 1X PBS containing 0.1% Triton® X-100 (cell permeabilization) for 5 minutes per wash by centrifugation at 1,500 rpm (332 rcf).

  1. Prepare Triton Washing Solution as follows:

    1X PBS 495 mL
    10% Triton X-100 5 mL
    1X PBS + 0.1% Triton X-100 500 mL

  2. Remove Fixing Solution (if using formaldehyde, collect in an appropriate waste container).

  3. Using a multi-channel pipettor, add 100 μL of Triton Washing Solution (room temperature (RT)). Add the solution down the sides of the wells carefully to avoid detaching the cells.

  4. Centrifuge at 1,500 rpm (332 rcf) for 5 minutes.

  5. Repeat washing steps 2 more times, removing wash manually each time.

    Do not allow cells to become dry during washing. Immediately add the next wash after manual disposal.

Block Cells

  1. Using a multi-channel pipettor, block cells/wells by adding 150 µL of Intercept® Blocking Buffer to each well. Add the solution by pipetting down the sides of the wells carefully to avoid detaching the cells.
  2. Allow blocking for 1 hour at RT with very gentle shaking on a plate shaker.

Primary Antibodies

Dilute Primary Antibodies

  1. Dilute the two primary antibodies in Intercept® Blocking Buffer. Combine the following antibodies for ERK target analysis:

    Rabbit anti-ERK antibody (1:200 dilution in the combined solution; Santa Cruz)

    Mouse anti-phospho-ERK antibody (1:100 dilution in the combined solution; Cell Signaling Technology)

  2. Mix the primary antibody solution thoroughly before adding to wells.

Incubate with Primary Antibodies

  1. Remove blocking buffer and add 50 µL of the desired primary antibody or antibodies in Intercept Blocking Buffer to cover the bottom of each well.
  2. Make sure to include control wells without primary antibody to serve as a source for background well intensity. Only add 50 µL of Intercept Blocking Buffer to control wells.
  3. Incubate with primary antibody for 2 hours with gentle shaking at RT.

Wash

  1. Prepare Tween® Washing Solution as follows:

    1X PBS 995 mL
    20% Tween 20 5 mL
    1X PBS + 0.1% Tween 20 1000 mL

  2. Remove primary antibody solution.

  3. Using a multi-channel pipettor, add 200 µL Tween Washing Solution (RT). Add solution down the sides of the wells carefully to avoid detaching the cells from the well bottom.

  4. Centrifuge at 1,500 rpm (332 rcf) for 5 minutes.

  5. Gently remove Tween Washing Solution by manually pipetting.

  6. Repeat washing steps 4 more times.

Secondary Antibodies

Dilute Secondary Antibodies

  1. Dilute the fluorescently-labeled secondary antibodies in Intercept® Blocking Buffer as specified below. To lower background, add Tween® 20 to the diluted antibody to a final concentration of 0.2%. Recommended dilution range is 1:200 – 1:1,200.

    Goat anti-Mouse IRDye® 680RD (1:800 dilution in the combined solution)

    Goat anti-Rabbit IRDye 800CW (1:800 dilution in the combined solution)

    Minimize exposure of the antibody vials to light.

  2. Mix the antibody solutions and add 50 µL of the secondary antibody solution to each well.

Incubate with Secondary Antibodies

  1. Incubate for 60 minutes with gentle shaking at RT. Protect plate from light during incubation.

    Use a large Western Incubation Box to protect plate from light during subsequent steps.

Wash

  1. Remove secondary antibody solution.
  2. Using a multi-channel pipettor, add 200 µL of Tween Washing Solution at RT (Wash). Add solution down the sides of the wells carefully to avoid detaching the cells from the well bottom.
  3. Centrifuge at 1,500 rpm (332 rcf) for 5 minutes.
  4. Gently remove Tween Washing Solution by manually pipetting.
  5. Repeat washing steps 4 more times. Protect plate from light during washing.

Image

  1. After final wash, remove wash solution completely from wells. Turn the plate upside down and tap or blot gently on paper towels to remove traces of wash buffer. For best results, scan plate immediately; plates may also be stored at 4 °C for several weeks (sealed and protected from light).
  2. Before plate scanning, clean the bottom plate surface and the Odyssey® Imager scanning bed (if applicable) with moist, lint-free tissue to avoid any obstructions during scanning.
  3. Scan plate with detection in both 700 and 800 nm channels.

Suggested Scan Settings

All settings may require adjustment for optimal data quality. Higher resolutions or scan qualities can be used, but the scan time will increase.

Instrument Resolution Scan Quality Intensity Setting (700 nm) Intensity Setting (800 nm)
Odyssey Classic 169 µM lowest 5 5
Odyssey DLx 169 µM lowest Auto Mode Auto Mode

Experimental Results

Figure 144. ERK activation in Jurkat, K-562, and THP-1 non-adherent cells in response to PMA as measured by specific antibody detecting dual-phosphorylated ERK (Thr202/Tyr204). The image represents a 96-well, two-color In‑Cell Western™ Assay with the 700 and 800 nm channels detecting phosphorylated and total ERK, respectively. The image was scanned using the Aerius™ Imager (similar to Odyssey® Sa) with scan setting of 200 μm resolution, focus offset of 3.5, and intensity of 3.5 (700 nm channel) and 5 (800 nm channel). Background (B) wells were incubated with secondary antibodies but no primary antibodies. The graph represents normalized quantitative data demonstrating the increase in ERK phosphorylation in response to PMA stimulation.

References

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