Troubleshooting Guide: Western Blot Figures

Figure 1 - Effect of Varying Primary Antibody Concentration:

Western blot optimization using R&D Systems rabbit anti-human/mouse/rat RSK1 affinity purified polyclonal antibody (Catalog # AF992). Human HeLa cells were solubilized in SDS gel buffer, and lysate from 1 x 10⁵ cells per lane was resolved by SDS-PAGE. Following electrophoresis, proteins were transferred to an Immobilon-P membrane (Millipore) and immunoblotted under the following conditions. A 30-second exposure to film is shown.

Bolded concentrations indicate the reduction of the primary antibody concentration.

1. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.15 M NaCl.
2. Blocked in 5% milk, 0.2 microgram/mL primary antibody in 5% milk and 0.15 M NaCl.

Figure 2 - Effect of Varying Primary Antibody Concentration, % Milk in Antibody Solution, and NaCl Concentration in Blotting Buffer:

Western blot optimization using R&D Systems rabbit anti-human PTP1B affinity-purified polyclonal antibody (Catalog # AF1366). Human HeLa (Lane 1), MCF-7 (Lane 2), and TF-1 (Lane 3) cells were solubilized in SDS gel sample buffer, and lysates from 1 x 10⁵ cells per lane were resolved by SDS-PAGE. Following electrophoresis, proteins were transferred to an Immobilon-P membrane (Millipore) and immunoblotted under the following conditions. A 30-second exposure to film is shown.

Figure 2a - Effect of Varying Primary Antibody Concentration in Antibody Solution with 2% Milk:

Bolded concentrations indicate the dilution of the primary antibody concentration.

1. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 2% milk and 0.15 M NaCl.
2. Blocked in 5% milk, 0.3 microgram/mL primary antibody in 2% milk and 0.15 M NaCl.
3. Blocked in 5% milk, 0.1 microgram/mL primary antibody in 2% milk and 0.15 M NaCl.

Figure 2b - Effect of Varying Primary Antibody Concentration in Antibody Solution with 5% Milk:

In addition to the dilution of the primary antibody concentration (bolded), the milk concentration in the Antibody Solution was also increased.

4. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.15 M NaCl.
5. Blocked in 5% milk, 0.3 microgram/mL primary antibody in 5% milk and 0.15 M NaCl.
6. Blocked in 5% milk, 0.1 microgram/mL primary antibody in 5% milk and 0.15 M NaCl.

Figure 2c - Effect of Varying NaCl Concentration in Blotting Buffer:

Bolded concentrations indicate the increased NaCl concentrations in the Blotting Buffer.

7. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.15 M NaCl.
8. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.5 M NaCl.

Figure 3 - Effect of Varying Milk and NaCl Concentrations:

Western blot optimization using R&D Systems rabbit anti-human PTP1B affinity-purified polyclonal antibody (Catalog # AF1366). Human HeLa (Lane 1), MCF-7 (Lane 2), and TF-1 (Lane 3) cells were solubilized in SDS gel sample buffer, and lysates from 1 x 10⁵ cells per lane were resolved by SDS-PAGE. Following electrophoresis, proteins were transferred to an Immobilon-P membrane (Millipore) and immunoblotted under the following conditions. A 30-second exposure to film is shown.

Bolded concentrations indicate adjustments in the milk and NaCl concentrations.

1. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 2% milk and 0.15 M NaCl.
2. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.15 M NaCl.
3. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.5 M NaCl.

Figure 4 - Effect of Varying NaCl Concentration in Blotting Buffer:

Western blot optimization using R&D Systems rabbit anti-human/mouse/rat RSK1 affinity purified polyclonal antibody (Catalog # AF992). Human HeLa cells were solubilized in SDS gel buffer, and lysate from 1 x 10⁵ cells per lane was resolved by SDS-PAGE. Following electrophoresis, proteins were transferred to an Immobilon-P membrane (Millipore) and immunoblotted under the following conditions. A 30-second exposure to film is shown.

Bolded concentrations indicate an increase in the NaCl concentration in the primary Antibody Solution.

1. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.15 M NaCl.
2. Blocked in 5% milk, 1.0 microgram/mL primary antibody in 5% milk and 0.5 M NaCl.