RPN2232, or Amersham ECL™ Select Western Blotting Detection Reagent, GE Healthcare, cat. Western blotting detection reagent (Amersham ECL™ Prime Western Blotting Detection Reagent, GE Healthcare, cat.Anti-mouse IgG–peroxidase antibody (Sigma, cat.Phosphate-buffered saline with Tween 20 (PBST see recipe).20× NuPAGE™ Transfer Buffer (Thermo Fisher Scientific, cat.Coomassie Brilliant Blue R-250 destain solution (see recipe).Coomassie Brilliant Blue R-250 stain solution (see recipe).NativeMark™ Unstained Protein Standard (Thermo Fisher Scientific, cat.NativePAGE™ 3-12% Bis-Tris Protein Gel, 1.0 mm, 10-well (Thermo Fisher Scientific, cat.20× NativePAGE™ Cathode Buffer Additive (Thermo Fisher Scientific, cat.20× NativePAGE™ Running Buffer (Thermo Fisher Scientific, cat.NativePAGE™ 5% G-250 Sample Additive (Thermo Fisher Scientific, cat.Native protein extraction buffer (see recipe make fresh), 4☌.Appropriate chemical for induction of gene expression and attendant protein expression.Deionized, distilled water (ddH 2O), with or without autoclaving.thaliana seeds (transgenic seeds expressing RPP7b-FLAG and HR4-HA proteins, referred to (Li et al., 2020)) We also describe two dimensional sodium dodecyl sulfide–polyacrylamide gel electrophoresis (2D SDS-PAGE), in which the protein molecules are separated according to molecular mass, as an alternative to direct western blotting for further protein-complex analyses. We use an intracellular immune receptor nucleotide-binding domain leucine-rich repeat (NLR) protein, RPP7, as an example to illustrate how one can monitor ligand-triggered protein oligomerization in plants by BN-PAGE (Li, Habring, Wang, & Weigel, 2020). In this protocol, we begin with a description of procedures for isolating proteins from transgenic Arabidopsis thaliana seedlings and analyzing them by BN-PAGE and direct western blotting. Combined with a downstream immunodetection approach, this enables determination of the molecular weights and oligomeric states of native protein complexes in plants. Based on their sizes, the protein molecules or complexes migrate to a position where the pore sizes of the gradient gel become restrictive. In addition, BN-PAGE can be employed to detect dynamic protein oligomerization when the protein complexes are relatively stable upon different treatments.īy taking advantage of Coomassie Brilliant Blue R-250 or G-250 dye to render a net negatively charged protein, BN-PAGE gel is able to separate proteins in their native conformations, regardless of the different isoelectric points of different proteins (Crichton, Harding, Ruprecht, Lee, & Kunji, 2013). Compared to gel filtration, which requires special equipment and large quantities of protein, BN-PAGE is much more sensitive and uses easily available conventional protein gel electrophoresis equipment, while at the same time requiring lower protein quantities. Gel filtration and blue native polyacrylamide gel electrophoresis (BN-PAGE) are the two principle approaches to studying native protein oligomerization in vitro and in vivo (Fiala, Schamel, & Blumenthal, 2011 see Current Protocols article Irvine, 2001 Wittig, Braun, & Schägger, 2006). Drain excess PBS/TBS from the membrane and transfer to appropriate enzyme substrate solution and incubate for time period recommended by manufacturer to visualize protein bands.Oligomerization of proteins controls numerous biochemical features, such as the stability of proteins and the activity of enzymes, immune receptors, and ion channels (Gell, Grant, & Mackay, 2012).Incubate the membrane with gentle agitation as follows: 4x5 minutes in wash buffer, followed by 2x5 minutes in PBS or TBS.(*No azide in the buffers if the secondary is labeled with HRP) Add appropriate enzyme conjugated secondary antibody diluted in wash buffer or blocking agent, and incubate for 1 hour at rT with gentle agitation in a sufficient volume to ensure coverage.Rinse the membrane in wash buffer (3x10 minutes), with gentle agitation and a sufficient volume to keep membrane well covered. Use a sufficient volume to keep the blot fully covered, with gentle agitation throughout. Incubate overnight at 4☌, or for 2 hours at room temperature. Incubate with primary antibody diluted in wash buffer or blocking buffer (an antibody concentration of 1-10 µg/ml is generally acceptable).
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