细胞功能测定

Proteins of the kinesin superfamily share a conserved motor domain, which both hydrolyses adenosine-5′-triphosphate (ATP) and binds microtubules. To determine the mechanism of action of a kinesin, it is necessary to relate the chemical cycle of ATP turnover to the mechanics of microtubu ...

The direct observation of single kinesins and microtubule-associated proteins (MAPs) has become a core tool for cytoskeleton research. We outline several variations to the core experiment that allow the researcher to explore structural and biophysical mechanisms underlying k ...

Biopolymers are essential for cellular organization. They bridge the cell interior, forming a framework that is used as a reference for different cellular organelles. This framework, called the cytoskeleton, is not static but constantly reorganizes. The dynamics of the cytoskeleton ...

Several microtubule-associated proteins localize in living cells selectively to an extended region at the growing microtubule plus ends. Over the last years, these plus-end-tracking proteins, also called +TIPs, have attracted considerable interest because they are involved in a ...

Microtubule organization and dynamics are controlled by a large set of cellular factors. An important group of microtubule regulators is microtubule plus-end-tracking proteins (+TIPs), which accumulate specifically at the growing microtubule ends, affect different phases of ...

Microtubules, polymers of the heterodimeric protein αβ-tubulin, give shape to cells and are the tracks for vesicle transport and chromosome segregation. In vitro assays to study microtubule functions and their regulation by microtubule-associated proteins require the availa ...

The fission yeast Schizosaccharomyces pombe is an attractive source of tubulin for biochemical experiments as it contains few tubulin isoforms and is amenable to genetic manipulation. We describe the preparation of milligram quantities of highly purified native tubulin from S. pom ...

Microtubules are cytoskeletal structures built of alpha- and beta-tubulins. Although tubulins are highly conserved throughout evolution, microtubules can be adapted to a range of different functions. A powerful mechanism that could regulate the functional specialization of ...

Alpha tubulin comprises a C-terminal tyrosine residue, which is subject to cyclic removal from the peptide chain by a still uncharacterized carboxypeptidase and re-addition to the chain by a tubulin tyrosine ligase. We have shown in different animal or human models that the presence or absen ...

Dynamic instability is a hallmark of the microtubule cytoskeleton. In order to regulate microtubule dynamics in vivo, a varied host of microtubule-associated proteins are mobilized to promote microtubule nucleation, growth, stabilization, catastrophe, depolymerization, ...

The centrosome is a unique organelle that nucleates and organizes the interphase microtubule array and facilitates bipolar spindle assembly during mitosis. Isolated centrosomes are ideal starting materials for biochemical and structural studies as well as for functional ass ...

Turbidity measurements are rapid and reliable methods to follow the effects of drugs, proteins, nucleotides, metals, and other factors on the assembly kinetics of tubulin into microtubules in vitro and have been in use since 1974. Improvements in spectrophotometers and software have re ...

Assaying microtubule dynamics in vitro requires stabilized nucleation centers, a method to immobilize individual microtubules onto a surface, and a specialized microscope to image the microtubule. Microtubules are polar structures with different dynamic properties at the p ...

Escherichia coli is a frequently used expression system for the generation of protein encoded by genes from diverse kingdoms and, thus, it is well suited for the production of protein antigens for antibody generation. It is a system of choice for many due to factors such as (1) the commercial availabi ...

Although immunoelectron microscopy is a powerful tool for visualizing the subcellular localization of target proteins, it is difficult to obtain and purify the specific antibodies required for this method. Instead of raising antibodies against individual target proteins, the u ...

Advances in genomic and proteomic platforms enable high-throughput studies for regulatory factors and interactors involved in signaling network at a molecular level. However, it has never been trivial to verify the omics data in vivo or functionally integrate the data in a cell signaling ...

Polyclonal antibodies are derived from multiple B-cell clones that have differentiated into antibody-producing plasma cells in response to an immunogen. Polyclonal antibodies raised against a single molecular species of antigen recognize multiple epitopes on a target molec ...

Before the advent of molecular methods to tag proteins, the visualization of proteins within cells by immunoelectron microscopy required the use of highly specific antibodies directed against the protein of interest. Thus, only proteins for which antibodies were available could be v ...

The detection of proteins with antibodies that are conjugated to gold particles has been a major asset to cell biology and the neurosciences, and knowledge about the subcellular location of antigens has formed the basis for many hypotheses regarding protein function. Many protocols have b ...

Monoclonal antibodies (mAb’s) have been used extensively in biochemical and biomedical studies, including immunoelectron microscopy. Production of mAb’s consists of four steps: immunizing the animal usually a mouse, obtaining immune cells from the spleen of the immunized mouse, f ...