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(Supplementary Table3): a rabbit polyclonal anti-Titin I103 (3.33 μg/mL), a mouse monoclonal anti-Titin T12 (2.5 μg/mL) (Boehringer Mannheim), a mouse monoclonal anti-α-actinin (1:4000) (EA-53, Sigma-Aldrich) antibody, and a guinea pig anti-Titin A165 antiserum (enzyme-...
The sigma cutoff used for the F2N data is 1.4 because of the high redundancy (33) and high CC(1/2) value (82%). Initial phases were obtained by molecular replacement by using the program Phaser72 and the coordinates of the designed structures as the search model. The high-resolution ...
Next the material on filters was washed twice with 80 μl of TE buffer, and then it was cleaved with 6 μg of DNAse (DN25, Sigma, St. Louis, MO) in 60 μl of 10 mm Tris-HCl, pH 7.8, containing 2.5 mm MgCl2 and 0.5 mm CaCl2 at 37 °C for 1 h. The obtained deoxynucleotide...
RNA was purified by collecting and transferring the TRIzol/chloroform upper aqueous phase to a new tube and the RNA precipitated with 750 μl of isopropanol (Sigma-Aldrich) for 10 min at 16,000g. RNA pellets were washed twice with 70% ethanol and solubilized in 200 μl of RNAse-...
Sigma-Aldrich MAB4418 Anti-Achaete Scute homolog 2 Antibody, clone 7E2 View Price and Availability Millipore ACK5003PA Amicon® Pro Affinity Concentration Kit Protein A with 3kDa Amicon® Ultra-0.5 Device View Price and Availability Millipore ACR5000NT Ni-NTA Reagent Kit View Price and Availab...
Aldolase, BSA, catalase, lysozyme, myosin, lactoferrin, carbonic anhydrase 2, and β-amylase were obtained from Sigma-Aldrich. RNase A was purchased from Thermo Fisher. Recombinant GST containing an N-terminal His tag was expressed in E. coli BL21 cells from the pDYH24 plasmid and purified wit...
The cleared supernatant subsequently mixed with 10 ml of equilibrated His-Select resin (Sigma-Aldrich) and incubated for 30 min at 4 °C with continuous mixing. The mixture was then loaded onto a column and washed with 300 ml wash buffer (50 mM Tris pH: 8.0, 2 M NaCl, ...
The upper part was incubated overnight with a mouse anti-PSD-95 (Millipore-Merck, MAB1596) 1/1000 and bottom part with a mouse anti-Histidine tag (Sigma-Aldrich, H1029) 1/1000. Following incubation with secondary antibodies, respectively, anti-mouse IRDye-680LT (LI-COR, 926–68020) (1...
The cells (at an OD600of two) were stained with mouse monoclonal anti-V5 (1/500; AbD Serotec, MCA2892) and rabbit polyclonal anti-FLAG (1/200; Sigma-Aldrich, F7425) in ice-cold staining buffer (wash buffer containing 0.5 mg ml−1BSA) for 1 h at 4 °C. They were the...