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SRPK2 generally never migrates at its predicted molecular weight (MW) due to post-translational modifications including, but not limited to, phosphorylation. The ~70 kDa band observed by the total antibodies is likely the C-terminal cleavage product. The SRPK2 protein is cleaved at D139 and D403, allowing the N-terminal fragments to travel to the nucleus. Please refer to Figure 3 in this article for more information. Notably, C-terminal phosphorylation of SRPK2 at T492 by Akt seems to prevent this cleavage, so it is uncommon to observe any of the lower MW fragments using a C-terminal phospho-specific SRPK2 antibody.
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When performing western blot using our COL1A1 antibodies, we detect the propeptide form of COL1A1, which is observed around 220kDa. The propeptide COL1A1 is processed to a mature form by cleavage of the N-terminal 1-161AA chain and the C-terminal 1219-1464AA portion resulting in a ~140kDa protein (UniProt ID P02452). This mature form may be detected by our COL1A1 antibodies when analyzing tissue samples but is not commonly observed in our hands in cell lysates.
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- Our Phospho-Smad1/5 (Ser463/465) (41D10) Rabbit mAb #9516 and Phospho-Smad1 (Ser463/465)/ Smad5 (Ser463/465)/ Smad9 (Ser465/467) (D5B10) Rabbit mAb #13820 are performance tested on extracts prepared from serum-starved cells treated with 50ng/ml BMP-2 for 30 minutes.
- 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 (http://www.cellsignal.com/products/buffers-dyes/phosphatase-inhibitor-cocktail-100x/5870) or Protease/Phosphatase Inhibitor Cocktail (100X) #5872 (
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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 Inhibit…