In-Cell Western Protocol

A. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

1. 10X Phosphate Buffered Saline (PBS): To prepare 1 L add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na₂HPO₄) and 2.4 g potassium phosphate, monobasic (KH₂PO₄) to 1 L dH₂O. Adjust pH to 7.4.
2. Formaldehyde, 16%, methanol-free, Polysciences, Inc. (cat# 18814), use fresh, store opened vials at 4°C in dark, dilute in PBS for use.
3. Blocking Buffer: To prepare 25 ml, add 2.5 ml 10X PBS, 1.25 ml normal serum from the same species as the secondary antibody (e.g., normal goat serum (#5425), normal donkey serum) and 21.25 ml dH₂O and mix well. While stirring, add 75 µL Triton X-100 (100%).
4. Antibody Dilution Buffer: To prepare 40 ml, add 4 ml 10X PBS to 36 ml dH₂O, mix. Add 0.4 g BSA and mix well. While stirring, add 120 µL Triton X-100 (100%).
5. Fluorochrome-conjugated secondary antibody.

NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.

B. Fixation and Immunolabeling

NOTE: Please refer to the immunofluorescence protocol for each antibody to identify the recommended antibody-specific fixation and permeabilization conditions.

NOTE: Cells should be grown, treated, fixed, and stained directly in multi-well plates.

NOTE: To avoid edge effects from uneven distribution of cells in individual wells, let newly-seeded plates stand at room temperature for one hour before placing in 37°C incubator (for more information, see Lundholt BK, Scudder KM, Pagliaro L. A simple technique for reducing edge effect in cell-based assays. J. Biomol. Screen. 2003 8, 566–70).

NOTE: To minimize false signal from scattered light and background fluorescence, use black-walled multi-well plates (e.g., BD Falcon, cat# 353948).

NOTE: Avoid touching the inside of the wells with pipettes or aspirators as this may leave an artifact that will affect subsequent quantification. Instead, empty wells by shaking inverted plate sharply over waste receptacle and then blotting with clean paper towel.

NOTE: Volumes indicated below are for use with standard 96-well plates. Adjust volume accordingly for plates with larger or smaller wells.

1. Grow and treat cells as desired in multi-well plates.
2. Fix cells with 50 µl 4% formaldehyde or 100% methanol, as recommended in the antibody-specific immunfluorescence protocol.
3. Allow cells to fix for 15 minutes at room temperature.
4. Shake inverted plate over aldehyde waste receptacle and blot with clean paper towel.
5. Rinse plate three times for 5 minutes each with room temperature PBS (100 µl/well).
6. Block cells in Blocking Buffer for one hour at room temperature (50 µl/well).
7. While blocking, prepare primary antibody by diluting in Antibody Dilution Buffer as indicated for Immunofluorescence (IF-IC) on datasheet.
8. Shake out blocking solution and add diluted primary antibody (total volume 50 µl/well).

NOTE: For double-labeling, prepare a cocktail of mouse and rabbit primary antibodies at their appropriate dilutions in Antibody Dilution Buffer.

9. Cover plate and incubate overnight at 4°C.
10. Rinse plate three times in PBS for 5 minutes each.
11. Add fluorochrome-conjugated secondary antibody* diluted in Antibody Dilution Buffer (total volume 50 µl/well) and incubate for one hour at room temperature in the dark.

NOTE: For double-labeling, prepare a cocktail of fluorochrome-conjugated anti-mouse and anti-rabbit secondary antibodies in Antibody Dilution Buffer.

12. Rinse plate three times in PBS for 5 minutes each.
13. To normalize for cell number in the absence of an antibody-based readout, label nuclei using DRAQ5 diluted 1:1000 in PBS (total volume 50 µl/well). Incubate at least 30 minutes at room temperature in the dark prior to scanning.

NOTE: DRAQ5 cannot be used in conjunction with DyLight 680 conjugated secondary antibodies. The emission spectra of DRAQ5 and DyLight 680 overlap and the signal produced by each dye cannot be distinguished.

NOTE: DRAQ5 can be emptied and the wells rinsed at least 1X with PBS for better optical resolution.

14. Scan plate according to manufacturer’s directions. (Be sure bottom of plate has been wiped clean prior to scanning).

*Recommended Secondary Antibodies:

Anti-Rabbit

• Anti-Rabbit IgG (H+L) (DyLight 680 Conjugate) #5366
• Anti-Rabbit IgG (H+L) (DyLight 800 Conjugate) #5151

Anti-Mouse

• Anti-Mouse IgG (H+L) (DyLight 680 Conjugate) #5470
• Anti-Mouse IgG (H+L) (DyLight 800 Conjugate) #5257