Publication protocol
rChrdl1 was titrated to identify the concentration that maximally impaired BMP4 and BMP7 (50 ng/ml) (catalog number 120-03; Peprotech). Total protein (30 to 40 μg) was separated by 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) and resolved proteins transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1 h with 5% milk solution, except for membranes used to detect pSmad1/5/8, total Smad2/3, and ShcA, for which 5% bovine serum albumin (BSA) solution was used as the blocking agent. Membranes were washed with 1× Tris-buffered saline (TBS)–1% Tween 20 and incubated overnight at 4°C using the following antibodies, as indicated: ShcA (1:1,000 dilution) (catalog number 610081; BD Biosciences), FLAG (M2) (1:1,000 dilution) (catalog number F1804; Sigma), ErbB2/Neu (C-18) (1:1,000 dilution) (catalog number sc-284-G; Santa Cruz), phospho-Smad 1/5/8 (1:1,000 dilution) (catalog number 9511; Cell Signaling), phospho-Smad1/8 (1:1,000 dilution) (catalog number 562508; BD Biosciences), Smad1 (1:1,000 dilution) (catalog number 38-5400; Invitrogen), Smad2/3 (1:1,000 dilution) (catalog number 610842; BD Biosciences), phospho-Smad2 (Ser465/467) (1:1,000 dilution) (catalog number 3101; Cell Signaling), and α-tubulin (1:5,000 dilution) (catalog number T9026; Sigma). Membranes were washed 3 times with 1× TBS–1% Tween 20 and incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000 dilution) for 1 h. The membranes were washed 3 times with 1× TBS–1% Tween 20 and visualized with an enhanced chemiluminescence system.
Full paper
Login or
join for free to view the full paper.
Videos
Check out videos that might be relevant for performing Western blotting Smad1 using SMAD1 Polyclonal Antibody from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.
We haven't found any additional videos for this experiment / product combination yet.