Bradford Assay Method The bradford dye-binding assay is a colorimetric assay for measuring total protein concentration. It involves the binding of Coomassie Brilliant blue to protein. There is no interference from cations nor from carbohydrates such as sucrose.
Detergents such as sodium dodecyl sulfate and triton x-100 can interfere with the assay, as well as strongly alkaline solutions.
Includes a general overview of the procedure and preparation of the standards in the protocol.
Cajal Body Isolation Protocol Cajal Body Isolation Protocol. Protocol includes: Sonication, Removal Nucleoi, Gradient One, Gradient two, Concentration and final enrichment of cajal bodies. Also includes: Making 2.55M sucrose stock and Analysis of the enriched Cajal body fraction.
CHO Centrosome Prep Protocol The key step is the lysis which solubilizes centrosomes away from nuclei by very low ionic strength lysis after treatment of cells with nocodazole and cytochalasin B. The released centrosomes are then centrifuged onto a Ficoll cushion (to avoid pelleting) and the interface between the lysate and the Ficoll is collected and the centrosomes are concentrated on a sucrose gradient. Fractions are assayed by spindown and double IF with 5051 serum and anti-tubulin and the pooled fractions are frozen...
Cryosectioning Protocol Protocol for cryosectioning. While the timing of the various steps in this protocol are probably not critical, process the tissue all at once to ensure that RNA and/or proteins do not get degraded. Includes: 20% Paraformaldehyde/4% Paraformaldehyde-PBS; Sucrose/PBS.
Equilibrium Density Gradient Percoll Protocol Cell fractionation of cellular components using Percoll a synthetic, colloidal solution of polyvinylpyrrolidone coated silica, specifically designed for sedimentation centrifugation. Percoll becomes a simple matter to establish a linear density gradient. Organelle separations are much easier to accomplish on Percoll density gradients than on sucrose gradients.
Extraction and Solubilization of Total Protein from Microorganisms Protocol The growth conditions of microbial cell cultures and the time of sample collection should be optimized and standardized when growing cells for protein extraction. Because cells may excrete proteases and other extracellular enzymes, and compounds in the medium may interfere with extraction, wash cultures with an isotonic buffer, such as PBS or sucrose before solubilization.
Gentle lysis of mammalian cells for cytochrome c release assay Lysis in the sucrose-containing Buffer ("Mito-Buffer") is supposed to prevent accidential disrupture of the mitochondria to prevent the leakage of mitochondrial proteins (such as cytochrome c) into the cytosol. Celldeath.de
Northern Blot Analysis of mRNA From Mammalian Polyribosomes Protocol The protocol consists of a method for the generation of cytoplasmic extracts from mammalian cells (in this case, 293T cells) without the disruption of polyribosomes, the separation of ribosomal components and polyribosomes by sucrose gradient centrifugation, the isolation of mRNA from these fractions, and detection of mRNA by Northern blot analysis.
