scientificprotocols

Author: Bart's Cookbook

This specific procedure was developed to assay the activity of the Lck kinase expressed from a retroviral vector in rat fibroblasts. You can obviously use fewer or more cells, depending on your needs. Don't skimp too much on the amount of S. aureus bugs that you use for precipitation or you will risk losses.

1. Immunoprecipitate the protein from 3 x 106 cells using 45 microliters S. aureus bugs. Resuspend the immunoprecipitates in 12 microliters kinase buffer (40 mM PIPES, pH 7.0, 10 mM MnCl2) at 4°C.

• The kinase reaction is carried out at room temperature in a total volume of 20 microliters that contains:
  • 15 microCi gamma-32P-ATP
  • 5 microliters immunoprecipitate
  • 2 mMolar [Val5]-angiotensin II
• Remove 5 microliter aliquots of the reaction mixture after 1, 3, and 5 minutes of reaction time. The incorporation of ATP is linear during this period.

scientificprotocols

Author: Hikmet Geckil's Lab

Abstract:

This is a fast, efficient and reproducible recovery procedure for periplasmic L-asparaginase from distinctly related gram-negative bacteria, Escherichia coli, Enterobacter aerogenes and Pseudomonas aeruginosa. As the method uses inexpensive organic solvent hexane and an aqueous salt solution, it is also highly cost-effective in comparison with the currently available techniques used for the release of this enzyme. As hexane is a highly water immiscible organic solvent, it can be removed easily from the top of the aqueous phase by a simple evaporation. Enzyme recoveries up to 3-fold with respect to sonication can be achieved with this system.

Method:

1. Harvest (10,000 rpm for 5 min) 24 h grown (in LB pH 7.5, 37 oC, 200 rpm) Escherichia coli, Enterobacter aerogenes or Pseudomonas aeruginosa cells.

• Wash once with 0.05 M potassium phosphate (KPi) bufferpH 8.6, and resuspended to A600= 5.0 in the same buffer with 1 % hexane.
• Incubate the suspenssion at roo

scientificprotocols

Authors: Mark Kamps and Tony Hunter

Method:

1. Label your protein with 32Pi. Then subject the protein to SDS polyacrylamide gel electrophoresis and transfer your gel-fractionated protein to Immobilon-P.

• Neither nitrocellulose nor nylon will work!
• Keep the membrane wet and wrap in Saran wrap.
• Add radioactive markings for orientation of film later
• Expose to film.
• Cut out band of interest, re-wet in methanol, rinse well in water and place in a screw-cap tube containing 150 µl 5.7 N HCl or enough to submerge your piece of membrane.

scientificprotocols

Author: Sara Lindgren

Purpose

The protocol describes how to prepare selenomethionyl (Se-Met) protein using a regular E.coli strain. Selenium can be used for phase determination in multi-wavelength anomalous diffraction (MAD) method. Se-Met can often replace methionine residues in a protein without affecting the protein's properties, therefore producing a protein advantageous for crystal structure solving. Also, the X-ray absorption edge of selenium is easily accessible by synchrotron radiation, making a Se-Met crystal ideal for collecting anomalous X-ray diffraction data. The Se-Met proteins can also be prepared using insect cells and CHO cells, which will be described in separate protocols.

Materials

1. LB media

• antibiotics (1000x conc.)

scientificprotocols

INTRODUCTION

Phosphate esters are widely distributed in any organism. Nucleic acids, metabolic intermediates like glucose-6-phosphate, energy-rich substrates (AMP, creatine phosphate) are some obvious examples. While many metabolic intermediates are activated through the transfer of phosphate groups (e.g., by kinases) it is equally important that phosphate esters can also be rapidly broken down. The hydrolytic removal of phosphate groups from phosphoesters is catalyzed by phosphatases. Many phosphatases are highly substrate-specific, like those enzymes involved in signal transduction. A number of phosphatases, however, cleave virtually any phosphate ester. Such unspecific enzymes function mainly in the catabolic breakdown of metabolites or nutrients. Depending on the pH at which such phosphatases have optimal activity, we distinguish between acidic phosphatases (also called acid phosphatases) and alkaline phosphatases. The latter enzymes require divalent metal ions as cofactors and are common in animal t

scientificprotocols

1. Aortas were removed from 6-week-old mice killed by CO2 asphyxiation and immediately transferred to a culture dish containing ice-cold endothelial cell basal medium (EGM-2; Cambrex Bio Science, Walkersville, MD).

• The periaortic fibroadipose tissue was carefully removed, paying special attention not to damage the aortic wall.
• 1 mm long aortic rings were sectioned and rinsed extensively in 8 consecutive washes of EGM-2.
• The rings were then individually embedded in 48-well plates previously coated with 50 µL synthetic basement membrane (Matrigel; BD Biosciences, Bedford, MA) per well.
• Next, an additional 50 µL of Matrigel was placed over each ring. After 1 hour, 500 µL EGM-2 was added to each well, and the cultures were incubated at 37°C for 5 days.
• The culture medium was changed on day 3 and the test compounds and vehicle were added.
• The aortic rings were photographed on day 5 at 4x magnification with an inverted microscope. For neovessel-regression experiments, the rings were cultured without dru

scientificprotocols

Authors: Ronald Frank and Stefan Dübel

This protocol was adapted from “Analysis of Protein Interactions with Immobilized Peptide Arrays Synthesized on Membrane Supports,” contributed by Ronald Frank and Stefan Dübel, Chapter 31, in Protein-Protein Interactions, 2nd edition (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

The following protocol describes the synthesis of short linear peptides, or peptide pools, on modified cellulose membranes, and the detection of their protein-binding partners. Peptides are synthesized from their carboxyl termini using Fmoc-amino acid derivatives. After completion of the synthesis and cleavage of all side-chain-protecting groups, the peptide array on the membrane is incubated with the potential interaction partners to identify their target sequences.

scientificprotocols

Authors: Britta Jedamzik and Christian R. Eckmann1

Corresponding author ([email protected]).

INTRODUCTION

RNA coimmunoprecipitation (co-IP) experiments are an extension of protein co-IP experiments in which in vivo RNA-protein complexes are investigated. This protocol describes how to perform RNA co-IPs from C. elegans whole-worm extracts. In principle, a protein-specific antibody is used to purify the protein of choice and its associated complex members from worm extract. This may also include RNA molecules associated with other protein components. To identify a specific mRNA molecule, all RNA molecules are first separated from the protein components after immunopurification. The mRNAs are then converted into cDNA by reverse transcription. Candidate mRNAs are detected by sensitive gene-specific amplification via polymerase chain reaction (PCR) in a semiquantitative manner. Since RNA molecules are very prone to degradation, it is crucial to avoid any kind of contamination with RNase activity in

scientificprotocols

Authors: Steven Raynard and Patrick Sung

Corresponding author ([email protected])

INTRODUCTION

Homologous recombination is an important mechanism for the repair of damaged chromosomes, for preventing the demise of damaged replication forks, and for several other aspects of chromosome metabolism and maintenance. The homologous recombination reaction is mediated by the Rad51 recombinase. In the presence of ATP, Rad51 polymerizes on single-stranded DNA (ssDNA) to form a nucleoprotein filament that is commonly referred to as the “presynaptic filament.” The presynaptic filament is capable of locating a homologous duplex DNA molecule and catalyzing invasion of the duplex to form a DNA displacement loop called the “D-loop.” This protocol describes an in vitro D-loop assay that uses a radiolabeled ssDNA oligonucleotide and a nonlabeled homologous supercoiled duplex DNA as substrates, and agarose gel electrophoresis together with PhosphorImaging for product analysis. To enhance

scientificprotocols

Authors: Vijay K. Tiwari and Stephen B. Baylin

Corresponding authors ([email protected]; [email protected])

INTRODUCTION

Progress in technologies to address long-range chromosomal interactions in vivo has extensively revised concepts about different aspects of transcriptional regulation. These methods allow probing physical proximities between chromatin elements without specifically identifying the protein components that mediate such interactions. Here we describe a detailed protocol for Combined 3C-ChIP-Cloning (6C) technology, which combines multiple techniques to identify the proteins that bridge distant genomic regions, while simultaneously identifying such physical proximities. This method is also useful for determining if a candidate protein might mediate long-range interactions, both in cis and in trans in the nucleus. We discuss how the 6C technique can be incorporated with other techniques to discover all the chromatin regions in the nucleus