How should I prepare my samples to detect phospho-Smad2/3?

Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb #3108, Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb #8828, Phospho-SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb #18338, and Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb #9520 are performance tested on extracts prepared from serum-starved cells that have been treated with 10ng/ml TGF-beta3 for 30 minutes.  It is possible that longer treatments (e.g., 24 hours) can lead to diminished signals for phospho-Smad2 and phospho-Smad3 because of Smad7 induction. 

​​Failure to include the appropriate serine/threonine phosphatase inhibitors can result in a diminished signal and/or inconsistent results between experiments. We always include sodium pyrophosphate (2.5mM final) and beta-glycerophosphate (1.0mM final) as serine/threonine phosphatase inhibitors in the lysis buffer for detection of the phospho-Smads. Our Phosphatase Inhibitor Cocktail (100X) #5870 or Protease/Phosphatase Inhibitor Cocktail (100X) #5872 can also be purchased as alternatives.

Sonication (15 seconds X 3) of the extracts is strongly recommended before any centrifugation in order to ensure maximal/consistent recovery of these targets (phospho-Smads are nuclear).

The total protein load should be at least 100ug per lane for detection of phospho-Smads in extracts prepared from whole tissue. A higher protein load for whole tissue extracts is necessary because only a portion of the cells would be expected to contain activated/phosphorylated Smad2/3. It is also important to note that the presence of detergent and/or reducing agent in your samples may lead to an overestimation of the protein load by Bradford assay. A more accurate protein concentration can be determined with a reducing agent/detergent compatible protein assay, such as Bio-Rad's RC DC Protein Assay (Catalog #5000120). The BSA standards should always be dissolved in the same buffer as the cell/tissue extracts. An alternative and probably easier approach would be to load at least 100ug per lane based on the actual weights of the tissue samples and not based on any protein assay.

Smad2/3 (TGF-β) extracts can be prepared as follows:

1. Grow cells (e.g., HT1080, HeLa, C2C12, NIH/3T3, KNRK, or C6) to 80-90% confluence.
2. Wash cells 1X with PBS.
3. Add appropriate serum-free media and incubate for 18 to 22 hours.
4. Treat cells with and without 10ng/ml Human Transforming Growth Factor ß3 (hTGF-ß3) #8425LC  for 30 minutes.
5. Wash cells 2X with ice-cold PBS.
6. Scrape cells into 1X Cell Lysis Buffer #9803  or any cell extraction buffer that includes appropriate phosphatase inhibitors.  Sodium pyrophosphate (2.5mM final) and beta-glycerophosphate (1.0mM final) should be included as serine/threonine phosphatase inhibitors in the cell extraction buffer. Phosphatase Inhibitor Cocktail (100X) #5870 or Protease/Phosphatase Inhibitor Cocktail (100X) #5872 can also be purchased as alternatives. Failure to include the appropriate serine/threonine phosphatase inhibitors can result in a loss of signal. 
7.  Sonicate for 15 seconds X3.  This step is essential for maximal/consistent recovery of the nuclear-localized phospho-Smad2/3.
8.  Microcentrifuge at 12,000rpm for 15-20 minutes.
9.  Add SDS Sample Buffer and heat at 95C for 3 minutes.  We recommend a total protein load of 20-30ug (200-300,000 cells) per lane.

Last updated: September 12, 2024