Cell Signaling Technology

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. 1X PBS/0.3% Triton X-100 (PBS/Triton): To prepare 1 L, add 100 ml 10X PBS to 900 ml dH2O. Add 3 ml Triton X-100 and mix.
  4. 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: This general fixation protocol will work with most antibodies and cell lines. However, we recommend trying different fixation methods to identify the optimal fixation protocol for each antibody and/or cell line.

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 multiwell plates (for example, BD Falcon, catalog #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. Treat cells as desired in multi-well plates.
  2. Add 100 µl 4% methanol-free formaldehyde in PBS to each well.
  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 (200 µl/well).
  6. Block cells in 5% normal serum from same species as secondary antibody (eg. normal goat serum, normal donkey serum) in PBS/Triton for one hour at room temperature (50 µl/well).
  7. Shake out blocking solution and add primary antibody diluting as indicated on datasheet in PBS/Triton (total volume 50 µl/well).

NOTE: For double-labeling, prepare a cocktail of mouse and rabbit primary antibodies at their appropriate dilutions in PBS/Triton.

  1. Cover plate and incubate overnight at 4°C or two hours at 37°C.
  2. Rinse plate three times in PBS for 5 minutes each.
  3. Incubate in fluorochrome-conjugated secondary antibody diluted 1:500 (Alexa Fluor® 680) or 1:800 (IRDye 800CW) in PBS/Triton for one hour at room temperature in dark.

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

  1. Rinse plate three times in PBS for 5 minutes each.
  2. Scan plate according to manufacturers directions. (Be sure bottom of plate has been wiped clean prior to scanning).

NOTE: For best optical resolution, shake out all PBS just before scanning plate.

posted November 2006

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