Example In-Cell Western Assay Development Project

Defining the Experimental System

These are some key components for the target assay. The target assay is further developed in the experiments in this section.

  • Model system

    • Target proteins: Total ERK1 and phosphorylated ERK1/2

    • Cells: HeLa cells (adherent cell line)

    • To stimulate ERK1/2 phosphorylation: TPA

    • To inhibit phosphorylation: U0126

  • Plate Format: Black-sided with clear well bottoms are needed.

    Plates used for this assay: Greiner Bio-One 96-Well µCLEAR® Plate, LICORbio PN 926‑19156 (8 pack) or 926‑19157 (32 pack)

  • Normalization method: CellTag™ 520 Stain (PN 926-41094)

  • Imaging system: (licor.com/bio/odyssey-m)

Choosing a Normalization Method and Determining Linearity

Cell Stain Linearity | In‑Cell Western Assay | pan-ERK1

The goal of this experiment is to determine the linear range of detection for the CellTag™ 520 Stain normalization method in the context of the target assay. The appropriate cell number should be within the linear range of detection and result in cells that are about 70 to 80 percent confluent at the time of detection.

CellTag 520 Stain has a broad linear range in this assay. In this assay, seeding 10,000 cells/well results in CellTag 520 signal that is within the linear range and results in cells that are approximately 80% confluent at the time of imaging. Comparable results were found for CellTag 700 Stain (data not shown). Using CellTag 520 Stain in the target In‑Cell Western Assay enabled simultaneous detection of pan-ERK1 in the 700 channel and p-ERK1/2 in the 800 channel.

Key finding: Seeding about 10,000 cells/well is an appropriate starting point for the assay being developed (Figure 122).

Experimental Details

  • Normalization method: CellTag 520 Stain (PN 926-41094)

  • Fixation/Permeabilization: 3.7% Formaldehyde + 0.2% Tween® 20

  • Blocking Buffer: Intercept® (PBS) Blocking Buffer (PN 927-70001)

  • Cells: HeLa Wild Type (WT) and HeLa ERK1 Knockout (KO1)

  • Acquisition Software: LI‑COR® Acquisition Software

  • Analysis software: Empiria Studio® Software with the [Preset] Cell Stain Linearity Plate Template

  • Imaging system: Odyssey M Imager

Results

Determining cell stain linearity using Empiria Studio
Figure 122. The cell number to seed was determined to be 10,000 cells/well for the target assay. Cell stain linearity was determined in the Empiria Studio® Software Cell Stain Linearity workflow. See In-Cell Western Assay Development Project for more information about determining cell stain linearity in Empiria Studio.

Validating Antibodies

The goal of the antibody validation experiments in this section is to ensure that chosen antibodies will work as needed in the target assay.

Antibody Validation | Western Blot | pan-ERK1

In this experiment, a primary antibody was evaluated in positive controls (HeLa WT cells) and negative controls (three stable clonal isolates of HeLa ERK1 KO cells).

Key finding: A single band is present in the positive control, but bands are not present in the negative control, providing evidence that the primary antibody will be specific to the target protein in the target assay (Figure 123).

Experimental Details

  • Target: pan-ERK1

  • Primary antibody: Human ERK1 (R&D Systems MAB1940)

  • Secondary Antibody: IRDye® 800CW Goat anti-Mouse IgG (926-32210)

  • Normalization method: Revert™ 700 Total Protein Stain (PN 926-11011)

  • Blocking buffer: Intercept® (PBS) Blocking Buffer (PN 927-70001)

  • Samples: HeLa whole cell lysates

  • Cells: WT and three stable ERK1 KO lines (knockout accomplished with SantaCruz CRISPR/Cas9 system)

  • Analysis Software: Empiria Studio® Software

  • Acquisition Software: LI‑COR® Acquisition Software

  • Imaging System: Odyssey M Imager

Results

Example Western blot antibody validation experimentExample fold change calculations performed in Empiria Studio
Figure 123. ERK1 KO samples did not show bands, providing evidence for antibody specificity. Three technical replicates of the experiment are shown. Western blot quantification, normalization, and fold change calculations were performed in Empiria Studio® Software.

Antibody Validation | In-Cell Western | pan-ERK1

In this experiment, a primary antibody was evaluated in positive controls (HeLa WT cells) and negative controls (three stable clonal isolates of HeLa ERK1 KO cells).

Key finding: Positive controls showed signal but negative controls did not, providing evidence that the primary antibody will be specific to the target protein in the target assay (Figure 124).

Experimental Details

  • Target: pan-ERK1

  • Primary antibody: Human ERK1 (R&D Systems MAB1940)

  • Secondary antibody: IRDye® 680RD Goat Anti-Mouse (PN 926-68070)

  • Normalization method: CellTag™ 520 Stain (PN 926-41094)

  • Fixation/Permeabilization: 3.7% Formaldehyde and 0.2% Tween® 20

  • Blocking buffer: Intercept® (PBS) Blocking Buffer (PN 927-70001)

  • Cells: HeLa WT and three stable HeLa ERK1 KO lines (knockout accomplished with SantaCruz CRISPR/Cas9 system PN)

  • Analysis Software: Empiria Studio® Software

  • Acquisition Software: LI‑COR® Acquisition Software

  • Imaging System: Odyssey M Imager

Results
Example In-Cell Western antibody validation experiment
Figure 124. ERK1 KO samples showed minimal signal, providing evidence for antibody specificity. Wells in the same column are technical replicates. In‑Cell Western Assay quantification, normalization, and fold change calculations were performed in Empiria Studio Software.

Choosing Fixation and Permeabilization Conditions

In these experiments, a variety of fixation and permeabilization combinations were evaluated via In‑Cell Western Assays.

Key finding: The same fixation and permeabilization conditions worked well for pan-ERK1 and p-ERK1/2, so those fixation and permeabilization conditions will be used in the target assay.

Fixation and Permeabilization Evaluation | In-Cell Western | pan-ERK1

In this experiment, fixation and permeabilization conditions were evaluated by determining the fold change between ERK1 signal detected in positive controls (HeLa WT) and ERK1 signal detected in negative controls (ERK1 KO) within different fixation/permeabilization conditions.

Key finding: Using 0.2% Tween® 20 for permeabilization and 4% formaldehyde for fixation yielded the largest fold change between positive and negative controls, indicating that 0.2% Tween 20 and 4% formaldehyde are acceptable conditions for the target assay (Figure 125).

Experimental Details

  • Primary antibody: Invitrogen – MA5-15896

  • Secondary antibody: IRDye® 800CW Goat Anti-Mouse (PN 926-32210)

  • Normalization method: CellTag™ 700 Stain (PN 926-41090)

  • Blocking buffer: Intercept® (PBS) Blocking Buffer (PN 927-70001)

  • Fixation/Permeabilization: 3.7% Formaldehyde + 0.1% Triton X100, 0.2% Tween 20, or 0.2% NP40; 100% MeOH, 90% EtOH, or 80% Acetone

  • Analysis software: Empiria Studio® Software using the [Preset] Fixation and Permeabilization Evaluation Plate Template

  • Acquisition Software: LI‑COR® Acquisition Software

  • Imaging system: Odyssey M Imager

Results
Evaluating fixation and permeabilization by In-Cell Western
Figure 125. Using 0.2% Tween® 20 for permeabilization and 4% formaldehyde for fixation yielded the largest fold change between ERK1 signal in positive controls and ERK1 signal in negative controls. Wells in the same column are technical replicates. In‑Cell Western Assay quantification, normalization, and fold change calculations were performed in Empiria Studio® Software. For fold change calculations, the KO1 values were set as the Control on the Set up charts page of the Fixation and Permeabilization Evaluation workflow.

Fixation and Permeabilization Evaluation | In-Cell Western | p-ERK1/2

In this experiment, fixation and permeabilization conditions were evaluated by determining the fold change between p-ERK1/2 signal detected in positive controls (TPA treated HeLa WT) and p-ERK1/2 signal detected in negative controls (U0126 treated HeLa WT) within different fixation/permeabilization conditions.

Key finding: Using 0.2% Tween® 20 for permeabilization and 4% formaldehyde for fixation yielded the largest fold change between positive and negative controls, indicating that 0.2% Tween 20 and 4% formaldehyde are acceptable conditions for the target assay (Figure 126).

Experimental Details

  • Primary antibody: CST-9101

  • Secondary antibody: IRDye® 800CW Goat Anti-Rabbit (PN 926-32211)

  • Normalization method: CellTag™ 700 Stain (PN 926-41090)

  • Blocking buffer: Intercept® (PBS) Blocking Buffer (PN 927-70001)

  • Fixation/Permeabilization: 3.7% Formaldehyde + 0.1% Triton X100, 0.2% Tween 20, or 0.2% NP40; 100% MeOH, 90% EtOH, or 80% Acetone

  • Analysis software: Empiria Studio® Software using the [Preset] Fixation and Permeabilization Evaluation Plate Template

  • Acquisition Software: LI‑COR® Acquisition Software

  • Imaging system: Odyssey M Imager

Results
Evaluating fixation and permeabilization by In-Cell Western
Figure 126. Using 0.2% Tween® 20 for permeabilization and 4% formaldehyde for fixation yielded the largest fold change between p-ERK1/2 signal in positive controls and p-ERK1/2 signal in negative controls. Wells in the same column are technical replicates. In‑Cell Western Assay quantification, normalization, and fold change calculations were performed in Empiria Studio® Software. For fold change calculations, the negative control values were set as the Control on the Set up charts page of the Fixation and Permeabilization Evaluation workflow.

Choosing a Blocking Buffer

In these experiments, a variety of blocking buffers were evaluated via In‑Cell Western Assay to determine the best blocking buffer for the target assay.

Key finding: The same blocking buffer performed well for pan-ERK1 detection and p-ERK1/2 detection, so it was chosen for use in the target assay.

Blocking Buffer Evaluation | In-Cell Western | p-ERK1/2

In this experiment, blocking buffers were evaluated by determining which blocking buffer was associated with the largest fold change difference for p-ERK1/2 signal detected in positive controls (TPA-treated HeLa WT) and p-ERK1/2 signal detected in negative controls (U0126-treated HeLa WT).

Key finding: Intercept® (PBS) Blocking Buffer showed the best fold change between positive and negative controls, so it makes sense to use as the blocking buffer for detecting p-ERK1/2 in the target assay (Figure 127).

Experimental Details

  • Target: p-ERK1/2

  • Primary antibody: CST – 9101

  • Secondary antibody: IRDye® 800CW Goat Anti-Rabbit (PN 926-32211)

  • Normalization method: CellTag™ 700 Stain (PN 926-41090)

  • Blocking buffer: Intercept (PBS) Blocking Buffer (PN 927-70001), Intercept (TBS) Blocking Buffer, Intercept (PBS) Protein-Free Blocking Buffer, Intercept (TBS) Protein-Free Blocking Buffer, 5% BSA PBS, 5% BSA TBS

    See licor.com/intercept for more information.

  • Fixation/Permeabilization: 3.7% Formaldehyde + 0.2% Tween 20

  • Analysis software: Empiria Studio® Software using the [Preset] Blocker Evaluation (Two Antibodies) Plate Template

  • Acquisition Software: LI‑COR® Acquisition Software

  • Imaging system: Odyssey M Imager

Results
Evaluating blocking buffer by In-Cell Western
Figure 127. Intercept® (PBS) Blocking Buffer showed the best fold change between positive and negative controls. There are four technical replicates of each sample. In‑Cell Western Assay quantification, normalization, and fold change calculations were performed in Empiria Studio® Software. For fold change calculations, the negative control values were set as the Control on the Set up charts page of the Blocker Evaluation workflow.

Blocking Buffer Evaluation | In-Cell Western | pan-ERK1 and p-ERK1/2

In this experiment, blocking buffers were evaluated by determining which blocking buffer was associated with the largest fold change difference between positive and negative controls. For pan-ERK1, positive controls were HeLa WT, and negative controls were HeLa KO1 cells. For p-ERK1/2, positive controls were TPA-treated HeLa WT and negative controls were U0126-treated HeLa WT.

Key finding:Intercept® (PBS) Blocking Buffer showed the best fold change between positive and negative controls for both antibodies, so it is the best from the options tested to use as the blocking buffer in the target assay (Figure 128).

Experimental Details

  • Target: pan-ERK1 and p-ERK1/2

  • Primary antibodies: Invitrogen MA5-15896 and CST - 9101

  • Secondary antibody: IRDye® 800CW Goat Anti-Rabbit (PN 926-32211) and IRDye 800CW Goat Anti-Mouse (PN 926-32210)

  • Normalization method: CellTag™ 700 Stain (PN 926-41090)

  • Blocking buffer: Intercept® (PBS) Blocking Buffer (PN 927-70001), Intercept (TBS) Blocking Buffer, Intercept (PBS) Protein-Free Blocking Buffer, Intercept (TBS) Protein-Free Blocking Buffer, 5% BSA PBS, 5% BSA TBS

    See licor.com/intercept for more information.

  • Fixation/Permeabilization: 3.7% Formaldehyde + 0.2% Tween® 20

  • Analysis software: Empiria Studio® Software using the [Preset] Blocker Evaluation (Two Antibodies) Plate Template

  • Acquisition Software: LI‑COR® Acquisition Software

  • Imaging system: Odyssey M Imager

Results
Evaluating blocking buffer by In-Cell Western
Figure 128. Intercept® (PBS) Blocking Buffer showed the best fold change between positive and negative controls for both antibodies. There are four technical replicates of each sample. In‑Cell Western Assay quantification, normalization, and fold change calculations were performed in Empiria Studio Software. For fold change calculations, the negative control values were set as the Control on the Set up charts page of the Blocker Evaluation workflow.

Determining Antibody Concentration

In these experiments, In‑Cell Western Assays were performed to determine the antibody concentration that provides the largest fold change between positive and negative controls, as well as minimal background.

Antibody Titration | In-Cell Western | pan-ERK1

In this experiment, six antibody concentrations were compared to see which would work best for the target assay. A fold change was calculated for pan-ERK1 signal detected in positive controls (HeLa WT) and negative controls (HeLa ERK1 KO1) in each of the six antibody concentrations. In general, larger fold changes are better, but other factors must also be considered.

Key finding: The antibody concentration 1:5,000 was chosen as the antibody concentration for the pan-ERK1 antibody (Figure 129).

The 1:5,400 antibody concentration option produced a large fold change and sufficient signal. Although the concentration 1:16,200 produced a larger fold change, the signal was too low.

Experimental Details

  • Target: pan-ERK1

  • Primary antibody: Human ERK1 (R&D Systems MAB1940)

  • Secondary antibody: IRDye® 800CW Goat Anti-Mouse (PN 926-32210)

  • Antibody concentrations: 1:100, 1:200, 1:600, 1:1800, 1:5400, 1:16200

  • Normalization method: CellTag™ 700 Stain (PN 926-41090)

  • Blocking buffer: Intercept® (PBS) Blocking Buffer (PN 927-70001)

  • Cells: HeLa WT and HeLa ERK1 KO

  • Analysis Software: Empiria Studio® Software with the [Preset] Antibody Titration (Two Antibodies) Plate Template

  • Acquisition Software: LI‑COR® Acquisition Software

  • Imaging System: Odyssey M Imager

Results
Performing antibody titration by In-Cell Western
Figure 129. The 1:5,400 antibody concentration option was chosen because it produced a large fold change and sufficient signal. There are four technical replicates of each sample. In‑Cell Western Assay quantification, normalization, and fold change calculations were performed in Empiria Studio® Software. For fold change calculations, the negative control values were set as the Control on the Set up charts page of the Antibody Titration workflow.

Antibody Titration | In-Cell Western | p-ERK1/2

In this experiment, six antibody concentrations were compared to see which would work best for the target assay. A fold change was calculated for p-ERK1/2 signal detected in positive controls (TPA-treated HeLa WT) and negative controls (U0126) in each of the six antibody concentrations. In general, larger fold changes are better, but other factors must also be considered.

Key finding: The 1:1,000 antibody concentration option was chosen, because it produced a large fold change and sufficient signal (Figure 130).

Experimental Details

  • Target: p-ERK1/2

  • Primary antibody 1: CST - 9101

  • Primary antibody 2: CST - 5726

  • Secondary antibody: IRDye® 800CW Goat Anti-Rabbit (PN 926-32211)

  • Antibody concentrations: 1:250, 1:500, 1:1000 (tested for both antibodies)

  • Normalization method: CellTag™ 700 Stain (PN 926-41090)

  • Blocking buffer: Intercept® (PBS) Blocking Buffer (PN 927-70001)

  • Cells: HeLa

  • Analysis Software: Empiria Studio® Software with the [Preset] Antibody Titration (Two Antibodies) Plate Template

  • Acquisition Software: LI‑COR® Acquisition Software

  • Imaging System: Odyssey M Imager

Results
Performing antibody titration by In-Cell Western
Figure 130. The 1:1,000 antibody concentration option was chosen, because it produced a large fold change and sufficient signal. There are four technical replicates of each sample. In‑Cell Western Assay quantification, normalization, and fold change calculations were performed in Empiria Studio Software. For fold change calculations, the negative control values were set as the Control on the Set up charts page of the Antibody Titration workflow.

Determining Z'-Factor

In this section, In‑Cell Western Assays were performed with the conditions chosen from previous assay development steps to calculate a Z'-Factor. The Z'-Factor indicates the robustness of the assay. See Z’-Factor Determination for more information.

Key finding: Assays for pan-ERK1 detection and p-ERK1/2 detection both yielded high Z'-Factor values that indicate a quality assay.

Z'-Factor Determination | In-Cell Western | pan-ERK1

Key finding: This assay was determined to have a Z'-Factor of 0.735, indicating it is a high quality assay (Figure 131).

Experimental Details

  • Target: pan-ERK1

  • Primary antibody: Human ERK1 (R&D Systems MAB1940)

  • Secondary antibody: IRDye® 680RD Goat Anti-Mouse (PN 926-68070)

  • Normalization method: CellTag™ 520 Stain (PN 926-41094)

  • Fixation/Permeabilization: 3.7% formaldehyde and 0.2% Tween® 20

  • Blocking buffer: Intercept® (PBS) Blocking Buffer (PN 927-70001)

  • Cells: HeLa

  • Positive control: HeLa WT

  • Negative control: ERK1 KO1

  • Analysis Software: Empiria Studio® Software with the [Preset] Z-Factor Determination 1

  • Acquisition Software: LI‑COR® Acquisition Software

  • Imaging System: Odyssey M Imager

Results
Determining Z'-factor by In-Cell Western
Figure 131. A Z'-Factor of 0.735 was determined, indicating a high quality assay. Wells in the same column are technical replicates. In‑Cell Western Assay quantification, normalization, and Z'-Factor calculations were performed in Empiria Studio® Software using the Z'-Factor Determination workflow.

Z'-Factor Determination | In-Cell Western | p-ERK1/2

Key finding: This assay was determined to have a Z'-Factor of 0.716, indicating it is a high quality assay (Figure 132).

Experimental Details

  • Target: p-ERK1/2

  • Primary antibody: CST-9101

  • Secondary Antibody: IRDye® 800CW Goat Anti-Rabbit IgG (PN 926-32211)

  • Normalization method: CellTag™ 700 Stain (PN 926-41090)

  • Fixation/Permeabilization: 3.7% formaldehyde and 0.2% Tween® 20

  • Blocking buffer: Intercept® (PBS) Blocking Buffer (PN 927-70001)

  • Cells: HeLa

  • Positive control: TPA-treated HeLa WT

  • Negative control: U0126-treated HeLa WT

  • Analysis Software: Empiria Studio® Software with the [Preset] Z-Factor Determination 2Plate Template

  • Acquisition Software: LI‑COR® Acquisition Software

  • Imaging System: Odyssey M Imager

Results

Determining Z'-factor by In-Cell Western
Figure 132. A Z'-Factor of 0.716 was determined, indicating a high quality assay. Wells in the same column are technical replicates. In‑Cell Western Assay quantification, normalization, and Z'-Factor calculations were performed in Empiria Studio® Software using the Z'-Factor Determination workflow.

Determining If Target Assays Meet the Goal

TPA Dose Response | In-Cell Western | pan-ERK1 and p-ERK1/2

This experiment is the first example of a target assay run using the conditions determined in the assay development experiments.

Key finding: EC50 values were calculated (Figure 133) that match expected results and closely match the EC50 calculated in the Western blot analog of this experiment (see TPA Dose Response | Western Blot | pan-ERK1 and p-ERK1/2).

Experimental Details

  • Targets: pan-ERK1 and p-ERK1/2

  • Primary antibodies: MAB-1940 and CST-1901

  • Normalization methods: CellTag™ 520 Stain (PN 926-41094) and pan-ERK1

  • TPA concentrations: 0, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 nM

  • Blocking buffer: Intercept® (PBS) Blocking Buffer (PN 927-70001)

  • Fixation/Permeabilization: 3.7% Formaldehyde and 0.2% Tween® 20

  • Analysis software: Empiria Studio® Software with the [Preset] Target Analysis Plate Template

  • Acquisition software: LI‑COR® Acquisition Software

  • Imaging system: Odyssey M Imager

Results
AB

Assessing dose response by In-Cell WesternAssessing dose response by In-Cell Western

CD

Assessing dose response by In-Cell WesternAssessing dose response by In-Cell Western

Figure 133. EC50 values were calculated that match expected results. A) The microwell plate is shown for this In‑Cell Western Assay with the amount of TPA used to treat wells shown in nM. Wells in the same column are technical replicates. B) In this chart, pan-ERK1 signal is normalized to CellTag™ 520 Stain, and the fold change for pan-ERK1 treatments are calculated with the 0 nM treatment as the denominator. The 0 nM treatment is set as the Control on the Set up charts page in the Empiria Studio® Software Target Analysis workflow. C) p-ERK1/2 signals are normalized to CellTag 520 Stain. The fold change is calculated the same way as described in part A. The EC50 value is 2.6nM (calculated outside of Empiria Studio Software). D) p-ERK1/2 signals are normalized to pan-ERK1. Fold change is calculated as described in part A. The EC50 value is 2.7 nM (calculated outside of Empiria Studio Software).

TPA Dose Response | Western Blot | pan-ERK1 and p-ERK1/2

This experiment is a Western blot analog to the In‑Cell Western Assay TPA Dose Response experiment.

Key finding: EC50 values were calculated (Figure 134) that match expected results and closely match the EC50 calculated in the In‑Cell Western Assay TPA Dose Response experiment (see TPA Dose Response | In-Cell Western | pan-ERK1 and p-ERK1/2).

Experimental Details

  • Targets: pan-ERK1 and p-ERK1/2

  • Primary antibodies: MAB-1940 and CST-1901

  • Normalization method: Revert™ 520 Total Protein Stain (PN 926-10011)

  • TPA concentrations: 0, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 nM

  • Blocking buffer: Intercept® (PBS) Blocking Buffer (PN 927-70001)

  • Analysis software: Empiria Studio® Software

  • Acquisition software: LI‑COR® Acquisition Software

  • Imaging system: Odyssey M Imager

Results
AB

Assessing dose response by 3-color Western blotAssessing dose response by 3-color Western blot

CD

Assessing dose response by 3-color Western blotAssessing dose response by 3-color Western blot

Figure 134. EC50 values were calculated that match expected results. A) Western blot images for this assay are shown. The 520 nm channel with Revert™ 520 Total Protein Stain is shown on the left, and the 700 and 800 nm channels with pan-ERK1 and p-ERK1/2 bands are shown overlaid on the right. B) In this chart, pan-ERK1 signal is normalized to Revert 520 Total Protein Stain, and the fold change for pan-ERK1 treatments are calculated with the 0 nM treatment as the denominator. The 0 nM treatment is set as the Control on the Set up charts page in the Empiria Studio® Software Western blot Target Analysis workflow. C) p-ERK1 signals are normalized to Revert 520 Total Protein Stain. The fold change for p-ERK1 is calculated using the same method described in part B for pan-ERK1. The EC50 value is 4.3nM (calculated outside of Empiria Studio Software). D) p-ERK2 signals are normalized to Revert 520 Total Protein Stain. The fold change for p-ERK2 is calculated using the same method described in part B for pan-ERK1. The EC50 value is 3.9 nM (calculated outside of Empiria Studio Software).

Measuring U0126 Inhibition | In-Cell Western | pan-ERK1 and p-ERK1/2

This experiment is an example Assay run to find the IC50 for U0126 inhibition using conditions from the assay development process.

Key finding: An IC50 was calculated in the low micromolar range (Figure 135), matching expectations.1

Experimental Details

  • Targets: pan-ERK1 and p-ERK1/2

  • Primary antibodies: MAB-1940 and CST-1901

  • Normalization methods: CellTag™ 520 Stain (PN 926-41094) and pan-ERK1

  • U0126 concentrations: 0, 0.156, 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 µM U0126 (200 nM TPA used to stimulate ERK1/2 phosphorylation)

  • Blocking buffer: Intercept® (PBS) Blocking Buffer (PN 927-70001)

  • Fixation/Permeabilization: 3.7% Formaldehyde and 0.2% Tween® 20

  • Analysis software: Empiria Studio® Software with the [Preset] Target Analysis Plate Template

  • Acquisition software: LI‑COR® Acquisition Software

  • Imaging system: Odyssey M Imager

Results
AB

Measuring U0126 inhibition by In-Cell WesternMeasuring U0126 inhibition by In-Cell Western

CD

Measuring U0126 inhibition by In-Cell WesternMeasuring U0126 inhibition by In-Cell Western

Figure 135. The calculated IC50 values match expected results. A) The microwell plate is shown for this In‑Cell Western Assay with the amount of U0126 used to treat wells shown in micromoles. Wells in the same column are technical replicates. B) In this chart, pan-ERK1 signal is normalized to CellTag™ 520 Stain, and the fold change for pan-ERK1 treatments are calculated with the 0 µM treatment as the denominator. The 0 µM treatment is set as the Control on the Set up charts page in the Empiria Studio® Software Target Analysis workflow. C) p-ERK1/2 signals are normalized to CellTag 520 Stain. The fold change for p-ERK1/2 is calculated using the same method described in part B for pan-ERK1. The IC50 value is 1.0 µM (calculated outside of Empiria Studio Software). D) p-ERK1/2 signals are normalized to pan-ERK1. The fold change for p-ERK1/2 is calculated using the same method described in part B for pan-ERK1 The IC50 value is 0.9 µM (calculated outside of Empiria Studio Software).

Conclusion

The assay development project reached its goal. Target assays were developed that can quantify changes in p-ERK1/2 expression, as expected.