|Storage buffer||10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.|
|Storage||Store at –20°C.|
1. Aspirate media from cultures and Wash the cells with 1X PBS. 2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice. 3. Sonicate for 10–15 sec to complete cell lysis and shear DNA. 4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice. 5. Centrifuge for 5 min (with Microcentrifuge). 6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins). NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer. 7. Electrotransfer to nitrocellulose/PVDF membrane.
Membrane Blocking and Antibody Incubations
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature. 2. Incubate the membrane in the blocking buffer for 1 hr at room temperature. 3. Wash three times for 5 min each with TBST.
b. Antibodies Incubation
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C. 2. Wash three times for 5 min each with TBST. 3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature. 4. Wash three times for 5 min each with TBST. 5. Proceed with detection.
Detection of Proteins
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST. 2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well. 3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
|Specificity||Smad1 Rabbit Recombinant mAb detects endogenous level of total Smad1.|
|Background||Smads are signal transducers for the members of the transforming growth factor-beta (TGF-beta) superfamily. Smads are subdivided into three subclasses based on their structure and function. Receptor-regulated Smads (R-Smads) have a C-terminal Ser-Ser-X-Ser motif, and are directly phosphorylated by the type I receptor kinases. R-Smads then form complexes with common-partner Smads (Co-Smads) and translocate into the nucleus, where they regulate the transcription of target genes. Inhibitory Smads (I-Smads) are induced by ligand stimulation and interfere with the receptor activation or complex formation of R-Smads. Upon BMP or TGFβ stimulation, cytosolic R-Smad members, including Smad1, Smad2, Smad3, Smad5 and Smad8, become phosphorylated by the type I receptors at the most C-terminal SSXS motif. Upon BMP or TGFβ stimulation, cytosolic R-Smad members, including Smad1, Smad2, Smad3, Smad5 and Smad8, become phosphorylated by the type I receptors at the most C-terminal SSXS motif. Although amino acid sequences of R-Smads share extremely high homology, Smad1/5/8 favor BMP signaling, while Smad2/3 mainly act downstream of the TGFβ pathway.|