For proteins that tend to precipitate, the addition of low concentrations of SDS (<0.01%) may be necessary. It should be noted that adding SDS to the transfer buffer may require optimization of other transfer parameters (e.g., time, current) to prevent over-t...
The transfer buffer used for electroblotting is similar to gel running buffer with the addition of methanol which helps proteins bind to the blot. Electrophoretic transfer can be accomplished under wet or semi-dry conditions. In a wet transfer, the gel/blotting paper/filter paper sandwich is ...
2. Soak filter papers and sponges in 1X Transfer Buffer for 10 minutes prior to assembly of the transfer “sandwich”. 3. After electrophoresis, remove the gel from the electrophoresis apparatus and equilibrate it by soaking in 1X Transfer Buffer for 10 minutes. 4. Prepare the transfer sandwi...
Protein ubiquitination regulates a wide range of cellular processes. The degree of protein ubiquitination is determined by the delicate balance between ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase (DUB)-mediated deubiquitination. In c
Precipitated proteins were then separated by centrifugation at 12,000× g for 30 min, washed with cold acetone, and re-suspended in a small volume of PBS Tris pH 8 buffer. 2.3. CyaA translocation assay Translocation of SopE-CyaA and SopA-CyaA into Int407 cells was assayed as described ...
Medium was poured off and cells were collected in NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0, 0.2% NP40/Igepal CA-630). Approximately 300 ml of buffer was used for 30 million cells. Lysates were clarified by centrifugation at 27,000 g for 15 min at 4 °C. ...
Fig. 1. Western blot analysis of CFm40L produced by a stable 293-based cell line. Aliquots of the purified protein were diluted with SDS sample buffer and divided into two tubes. One tube was heated at 96°C, and the other was left unheated. Both boiled (B) and unboiled (UB) sampl...
Western blot analysis was performed as described previously [12]. Briefly, the supernatant was concentrated by super filter (5 kDa, Minipore) and mixed with an equal volume of sodium dodecyl sulfate (SDS) sample buffer. The proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) ...
Optimization of BRET by circularly permuted Venus In order to further enhance the luminescence intensity, we attempted to optimize the spatial arrangement of the donor and acceptor by using circularly permuted Venus variants (cp50Venus, cp157Venus, cp173Venus, cp195Venus and cp229Venus)18,19. The...
Samples were loaded onto 12.5% native poly- acrylamide gels and electrophoresis was performed at 100 V and 4 °C in common SDS-PAGE running buffer containing 0.1% SDS for 2.5–4 hours. TraH was probed by western blotting as described for the protease protec- tion assay. NMR ...