Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability
[Abstract] Stopped-Flow Light Scattering (SFLS) is a method devised to analyze the kinetics of fast chemical reactions that result in a significant change of the average molecular weight and/or in the shape of the reaction substrates. Several modifications of the original stopped-flow system have been made leading to a significant extension of its technical ...
Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp
[Abstract] This protocol describes the isolation of tonoplast vesicles from tomato fruit. The vesicles isolated using this procedure are of sufficiently high purity for downstream proteomic analysis whilst remaining transport competent for functional assays. The methodology was used to study the transport of amino acids during tomato fruit ripening (Snowden ...
Preparation of Golgi Membranes from Rat Liver
[Abstract] This protocol details the isolation of enriched Golgi membranes from rat liver, using discontinuous density gradient centrifugation. This high-yield extraction method is useful for several applications, including immunoprecipitation of solubilised Golgi membrane proteins (preparation included) and electron microscopy. Protocol adapted from ...
Ciliary and Flagellar Membrane Vesicle (Ectosome) Purification
Author: William Dentler,
date: 06/20/2014,
view: 8414,
Q&A: 0
[Abstract] Eukaryotic cilia/flagella are ideal organelles for the analysis of membrane trafficking, membrane assembly, and the functions of a variety of signal transduction molecules. Cilia are peninsular organelles and the membrane lipids, membrane proteins, and microtubular-associated components are selectively transported into cilia through the region ...
Purification of HCV-remodeled and Control ER Membranes
Authors: David Paul and
Ralf Bartenschlager,
date: 05/20/2014,
view: 6538,
Q&A: 0
[Abstract] As for all positive strand RNA viruses, hepatitis C virus (HCV) RNA replication is tightly associated with rearranged host cell membranes, termed viral replication factories. However, up to now little is known about both viral and cellular constituents of viral replication factories. Here, we describe a protocol to specifically isolate ...
Preparation of Bacillus subtilis Cell Lysates and Membranes
Authors: Juri Niño Bach and
Marc Bramkamp,
date: 01/20/2014,
view: 13997,
Q&A: 0
[Abstract] A common feature of every eukaryotic and prokaryotic cell is that they exhibit a plasma membrane. In Bacillus subtilis (B. subtilis) roughly 25% of all proteins are putative trans- or membrane associated proteins. Here we describe a relatively simple method to separate and prepare membrane and cytosolic proteins by ...
β1 Integrin Cell-surface Immunoprecipitation (Selective Immunoprecipitation)
Author: Ralph T. Böttcher,
date: 11/05/2013,
view: 9496,
Q&A: 0
[Abstract] Immunoprecipitation (IP) is a widely used method to isolate a specific protein from a mixed protein sample using an antibody that exclusively binds to that particular protein. This technique allows studying protein-protein and protein-nucleic acid interactions or to identify post-translational protein modifications. Many proteins, in particular ...
Isolate and Sub-fractionate Cell Membranes from Caulobacter crescentus
Author: Khatira Anwari,
date: 10/20/2012,
view: 11774,
Q&A: 1
[Abstract] Cell membranes from Caulobacter can be isolated and separated into inner and outer membranes according their characteristic buoyant densities on a sucrose gradient. Fractionation can be used to determine the localisation of uncharacterised proteins and to enrich protein complexes present in either of these membranes for biochemical ...