lysate Protocols

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

ANTIBODY PURIFICATION by affinity chromatography. By Beth, Mullins Lab UCSF. To affinity purify antibodies, generate lots of E. coli lysate that contains your antigen. If the protein can stand freeze thawing, then go ahead and purify the protein from e. coli lysate and keep it frozen until you need to couple it to a CH-sepharose column. - [Read ANTIBODY PURIFICATION by affinity chromatography]

Category: Protein Protocols: Protein Extraction From Cells

Cell Lysate Extracts. Great protocols for cell lysis preparation from a variety of cell types. There are numerous methods of cell stimulation and lysis. For a given protein, Upstate’s Laboratories determine the specific treatment upon initial testing of its products. It is important to select the correct cell line, stimulation procedure (if any), and lysis protocol. Upstate. - [Read Cell Lysate Extracts]

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 Organelle Protocols: Subcellular Fractionation Protocols

Fractionation of (a) vacuolar and subvacuolar vesicles and (b) vacuole and cytoplasm-to-vacuole targeting (Cvt) vesicles from yeast spheroplasts in a pre-formed discontinuous iodixanol gradients. Protocol includes: Formation of yeast spheroplasts; Isolation and vesiculation of the vacuoles; Separation of the vacuolar and subvacuolar vesicles; Separation of vacuoles and Cvt vesicles from a yeast spheroplast lysate. - [Read Fractionation of Vacuolar and Subvacuolar vesicles and Vacuole and Cytoplasm-to-Vacuole Targeting]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

A crude lysate gel assay can be performed to roughly quantitate the DNA in lysates. This is often a valuable time saving step to determine if the phage yield is sufficient to warrant continuing the procedure. - [Read Gel Assay to Determine DNA Content of Phage Lysates Protocol]

Category: Pharmacology and Toxicology Protocols: Drug Discovery

To determine the selectivity of the inhibitors identified during screening efforts we developed gel-elongation assay using crude bacterial lysate directly to determine the target specificities of fatty acid synthesis inhibitors. - [Read Gel Elongation Assay for Type II Fatty Acid Synthesis Protocol]

Category: Lipid Protocols

New screening efforts and chemical modifications of existing compounds have been attempted to identify more selective and potent inhibitors. To determine the selectivity of the inhibitors identified during screening efforts we developed gel-elongation assay using crude bacterial lysate directly to determine the target specificities of fatty acid synthesis inhibitors. - [Read Gel-elongation Assay for Type II Fatty Acid Synthesis Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

There are essentially three parts to this protocol: 1. growth of at least 5x10e8 pfu phage to provide an inoculum growth of a larger liquid lysate that will produce about 5x10e12 pfu; 2. concentration and purification of the phage, and; 3. DNA preparation. - [Read Growth and Purification of 25-100 ug Lambda Clone DNA Protocol]

Category: Immunology: Immunoprecipitation Protocols

Protocol for immunoprecipitation of mRNA-protein complexes. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. - [Read Immunoprecipitation of mRNA-Protein Complexes Protocol]

Category: Immunology: Immunoprecipitation Protocols

Protocol for immunoprecipitation. Includes: Cell Lysate Preparation; Cell Lysate Preclearing; Immunoprecipitation; Immunoprecipitation Buffers. - [Read Immunoprecipitation Protocol]

Category: Protein Protocols: Immunoprecipitation Protocols

Immunoprecipitation Protocol and Information. IP. Cell Lysate Preparation, preclearing, Immunoprecipitation Buffers. Ebioscience. - [Read Immunoprecipitation Protocol and Information]

Category: Immunology: Immunoprecipitation Protocols

Protocol for immunoprecipitation. Includes: Preparing Cell Lysate; Immunoprecipitating; Western Blotting. - [Read Immunoprecipitation Protocol II]

Category: Bacterial Cell Culture Protocols

In this protocol, bacterial cells are lysed by being subjected to short, intense treatments with ultrasound, which breaks the cell walls and shears the DNA into sizes that will not affect the viscosity of the samples. Note that this method causes some denaturation of the samples. The resulting lysate is ready for preclearing. - [Read Immunoprecipitation: Lysing Bacteria by Sonication Protocol]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

To reduce backgrounds and to improve the signal-to-noise ratio, an antibody that does not recognize the antigen being studied can be added to the lysate and processed as for a normal immunoprecipitation. Any nonspecific proteins that might contaminate the final immunoprecipitation step are presumably removed with this irrelevant antibody. - [Read Immunoprecipitation: Preclearing the Lysate Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol for in vitro transcription and translation using the coupled reticulocyte lysate system. This protocol is designed to test random samples on a protein gel. Scale up the reactions accordingly. Protocol includes: Procedure, Solutions, BioReagents and Chemicals and protocol hints. - [Read In Vitro Transcription and Translation Using the Coupled Reticulocyte Lysate System]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol for the isolation of infectious HIV-1. Includes: HIV-1 capture strategy; Protocol for the isolation of HIV-1 virions; Magnetic labeling; Magnetic separation; Elution option A for virion lysate; Elution option B for intact virions. - [Read Isolation of Infectious HIV-1 Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

5 ml liquid lysates are prepared when a small amount of DNA from a large number of lambda clones is needed. The lysates can be made using 10- 20 ul of a stock lysate or a 100-fold amplified phage "macroplaque" as the inoculum. - [Read Liquid Phage Lysates Protocol]

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

No special treatment is required to prepare a lysate for the active collection. The following procedure should be used for long-term storage of lambda clones in the archival collections. The phage are diluted in media containing 7% DMSO and frozen at -80 degrees C. - [Read Long Term Lambda Phage Storage Protocol]

Category: Protein Microarray Protocols

Lysate Microarrays. High throughput methods that detect abundance and post-translational modification. MacBeath Lab - [Read Lysate Microarrays - Information]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Protocol for mini-prep. Includes: PREPARATION OF E. COLI; PRODUCTION OF CLEAR LYSATE; cDNA ISOLATION. - [Read Mini-Prep Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol for preparation of messenger-dependent rabbit reticulocyte lysate. Informamtion also added to test that the lysate is working before doing the Nuclease inactivation - [Read Preparation of Messenger-Dependent Rabbit Reticulocyte Lysate]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol for the preparation of rabbit reticulocyte lysate cell-free system. Protocol includes information on: Solutions; BioReagents and Chemicals and Protcol Hints. - [Read Preparation of Rabbit Reticulocyte Lysate Cell-Free System Protocol]

Category: C. elegans Protocols

Protocol was designed to rapidly generate small scale cytosolic extracts of C. elegans for Western or IP (has not been tested for RNA work). The protocol works well for between 50 to 5000 worms and has not been extensively tested on larger a scale, though it should work. Includes: Collection; Sonication; Clearing lysate; Immunoprecipitation. - [Read Preparation of Worm Extracts by Sonication Protocol]

Category: Yeast Protocols: Yeast Protein Protocols: Yeast Immunoprecipitation Protocols

Protocol for protein immunoprecipitation from a Yeast cell lysate. - [Read Protein Immunoprecipitation from a Yeast Cell Lysate Protocol]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Protocol for luciferase assay for in vitro detection. Protocol includes: Before cell lysate preparation; Cell lysate preparation; Protocol for manual luminometers; Protocol for plate reading luminometer; Preparation of protein assay reagent; Protein standards. - [Read Protocol for Luciferase Assay for In Vitro Detection]