ionic Protocols

Category: Cell Organelle Protocols: Nucleus Protocols

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... - [Read CHO Centrosome Prep Protocol]

Category: Cell Culture Protocols: Cell Lysis Protocols

For cells grown in tissue culture, the most useful method of lysis is treating with detergents, as described in this protocol. Non-ionic detergents, such as NP-40, solubilize the plasma and intracellular membranes, break many weak intermolecular bonds, and solubilize most of the commonly studied protein antigens. RIPA lysis buffer may be used as a more rigorous extraction buffer to release all but the insoluble proteins of the cell and to break most weak noncovalent interactions. - [Read Lysing Tissue-Culture Cells for Immunoprecipitation Protocol]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol for the optimization of absorption condition for dye-ligand affinity chromotography. Generally, low pH and low ionic strength, absence of phosphate ions, and the presence of divalent metals ions increase the binding of proteins to immobilized triazine dyes. - [Read Optimization of Adsorption Conditions for Dye-Ligand Affinity Chromatography Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

Bacteriophage {lambda} particles are recovered from bacterial lysates by precipitation with polyethylene glycol at high ionic strength. - [Read Precipitation of Bacteriophage {lambda} Particles from Large-scale Lysates Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Bacteriophage M13 single-stranded DNA is prepared from virus particles secreted by infected cells into the surrounding medium. The filamentous particles are concentrated by precipitation from a high-ionic-strength buffer with polyethylene glycol. Subsequent extraction with phenol releases the single-stranded DNA, which is then collected by precipitation with ethanol. This protocol is generally used to prepare single-stranded DNA from a small number of M13 isolates. - [Read Preparation of Single-stranded Bacteriophage M13 DNA Protocol]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for the purification of DNA recovered from agarose and polyacrylamide gels by anion-exchange chromatography. Fragments of DNA recovered from agarose gels are sometimes poor templates or substrates in subsequent enzymatic reactions.  This problem can be solved by binding the DNA to a positively charged matrix, such as DEAE-Sephadex or DEAE-Sephacel, in buffers of low ionic strength.  After washing the matrix, the DNA is eluted by raising the strength of the buffer. - [Read Purification of DNA Recovered Anion-exchange Chromatography Protocol]

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

When many RNA samples are to be processed or when working with small amounts (<50 µg) of total mammalian RNA, the technique of choice is batch chromatography on oligo(dT)-cellulose. The method described in this protocol uses a combination of temperature and ionic strength to maximize binding and recovery of polyadenylated RNA. IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O. Joseph Sambrook and David W. Russell. - [Read Selection of Poly(A)+ RNA by Batch Chromatography - Subscription Required]

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Electrocompetent bacteria are prepared by growing cultures to mid-log phase, washing the bacteria extensively at low temperature, and then resuspending them in a solution of low ionic strength containing glycerol. DNA is introduced during exposure of the bacteria to a short high-voltage electrical discharge.