Tla 55 Rotor, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 96/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae"
Article Title: Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae
Journal: Molecular Biology of the Cell
Figure Legend Snippet: Y14D mutant readily dissociates into monomers in nonboiled samples, shifts the distribution toward monomers on sucrose gradients, and partitions into the cytosolic fraction. (A) Western blot analysis of boiled and nonboiled lysates of Myc-tagged Cav1 constructs in Cav1 − / − MLECs. To detect Cav1 − / − MLECs expressing WT-Cav1, Y14F, Y14D and C156S mutants were lysed and blotted using standard conditions (including RIPA buffer and boiling) or gently solubilized in saline buffer supplemented with 2% ODG and heated at 60°C to preserve higher- order oligomers (“nonboiled”). Membranes were blotted with anti-Cav1 pAb. HEK cells expressing (B) WT-Cav1, (C) Y14F-Cav1, or (D) Y14D-Cav1 were fractionated by density gradient centrifugation on 5–30% sucrose gradients. Eleven equal fractions, with the top fraction (1) being the lightest and bottom fraction (11) the heaviest, were analyzed by Western blot using anti–total Cav1 pAb. Bar graphs depicting the distribution (relative abundance) of Cav1 oligomers and monomers in each lane were calculated as a percentage of the total Cav1 in the entire blot. (E) Analysis of cytosolic and total membrane fractions from Cav1 − / − MLECs expressing Myc-tagged Cav1 cDNAs. Detergent-free cell homogenates were clarified by low-speed spin (500 × g ) to remove nuclei and unbroken cells, and then supernatants were ultracentrifuged at 55,000 rpm in a TLA-55 Beckman rotor to separate supernatant (cytosol/microvesicle fraction) from the pelleted total membrane fraction (pellet). The two fractions were then solubilized and adjusted to the same concentration, heated in sample buffer at 60°C (“nonboiled”), and resolved on SDS–PAGE gels, followed by detection with anti–total Cav1 pAb. The C156S Cav1 mutant was used as a negative control for Cav1 oligomerization ( Bakhshi et al ., 2013 ).
Techniques Used: Mutagenesis, Western Blot, Construct, Expressing, Gradient Centrifugation, Concentration Assay, SDS Page, Negative Control