Popular loading controls for cytoplasmic proteins include housekeeping proteins, such as beta-actin or beta-tubulin for nuclear proteins, histone H1 or histone H3 are commonly used. Select the optimal loading control based on the cellular localization of your protein. Treatment with the proteasome inhibitor MG132 also preserves ubiquitylation and blocks protein degradation by the 26S proteasome ( Kisselev et al. However, up to tenfold higher concentrations are crucial to maintain ubiquitylation of certain proteins, such as IRAK1 ( Emmerich and Cohen 2015). IAA and NEM are commonly used at a concentration of 5–10 mM. For this reason, it is essential to add EDTA or EGTA as well as iodoacetamide (IAA) or N-ethylmaleimide (NEM) to the cell lysis buffer. Removal of ubiquitin conjugates can also easily occur by protein ubiquitin hydrolases, known as deubiquitylases (DUBs). Therefore, to conserve SUMOylation, the addition of isopeptidase inhibitors to the lysis buffer is recommended ( Xiao et al. This is due to the activity of isopeptidases that remove the SUMO conjugates and that often only a small fraction of a protein is SUMOylated. The identification of SUMOylated proteins can be particularly challenging. Common protease inhibitors include phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, and pepstatin. However, these conditions could affect the integrity of certain proteins therefore lysis buffers are routinely supplemented with protease inhibitor cocktails. To limit protease activity, strong denaturing agents, like urea, can be added to the buffer. Inhibit protein degradation and conserve post-translational modifications during sample preparationĭisruption of cell membranes releases enzymes that can degrade proteins despite minimizing their activity by keeping the samples at low temperature ( Scopes 1994). Several protein extraction protocols, such as trichloroacetic acid/acetone precipitation method or phenol-based extraction, have been developed for plant tissues ( Wang et al. The extraction process has to be optimized for the specific plant tissue you are using. Extraction of proteins from plant tissues is particularly challenging as they are rich in proteases and contain high levels of metabolites that can interfere with protein extraction ( Wang et al. More information on this topic can be found in our Five Tips for Picking the Right Cell Disruption Method for Protein Analysis video.Īs lipids can impact the quality of your western blots, ensure you remove fat from your samples. Ensure that you have optimized the power settings on mechanical disruptors and use pre-chilled equipment. It is important to avoid foaming during this step as it may decrease your recovery volume. To further dissociate the tissue and to shear cellular DNA, sonication of samples might be needed. Harsher disruption methods, like homogenization techniques, are required for tissues due to the higher degree of sample structure compared to cells.
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