Buffers and Reagents

Ultrapure water (18.2 MS2 cm), was obtained from a MiUipore water purification system and was used for preparation of sample solutions. Analytical Grade Acetonitrile (MeCN) (Fischer Scientific) and Trifluoroacetic Acid (TEA) (Sigma-Aldrich) were used for all ZipTip buffers (see Sect. Denaturing MS— ZipTip Buffers ). The coenzyme A derivatives (acetyl-, butyryl- and malonyl) used in assays were purchased from Sigma-Aldrich. iV-acetylcysteamine (SNAC) thioesters were provided by the Piel lab (ETH, Zurich), and stock solutions were solubilised in dimethyl sulfoxide (DMSO) (Fischer Scientific). lodoacetamide (Sigma-Aldrich) was used for all alkylation reactions. 

All expression vectors were supplied by Professor Jom Piel (ETH, Zurich). Each vector contains the cDNA sequence for the protein domain of interest. Expression conditions are detailed in Table 2.1. 

All reagents in this section were purchased from Fischer Scientific or Sigma Aldrich. The compositions of buffers used for various aspects of protein purification are fisted below  

Re-suspension/Binding Buffer 25 mM Tris HCl, 500 mM NaCl, pH 7.6 Eluting Buffer 25 mM Tris HCl, 500 mM NaCl, 500 mM Imidazole, pH 7.6 Stripping Buffer 25 mM Tris HCl, 500 mM NaCl, 50 mM EDTA, pH 7.6 Nickel Sulphate 100 mM NiS04 

SDS-PAGE Running Buffer 25 mM Tris HCl, 192 mM Glycine, 0.1 % SDS TAE Buffer 40 mM Tris HCl, 20 mM Acetic acid, 1 mM EDTA 

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The following protocol assumes that the user has at least two pure proteins (or biomolecules) of known sequences, which are able to interact specifically in solution. One of the two proteins (the prey) is end labeled with a fusion tag or another detectable component and the other protein contains at least one thiol group. All buffers and reagents used in this protocol should be of high purity and contain a very low metal content to prevent nonspecific cleavage reactions. 

The primary advantage of the Coomassie plus protein assay is that it is generally compatible with most of the buffers and reagents found in samples and is unaffected by the presence of chelating agents, reducing agents, or free sulf-hydryls in the sample. 

This section describes the preparation of buffers and reagents used in the manipulation of nucleic acids. 

The mass spectrometer detectors place new demands on the HPLC system. The MS interface requires use of volatile buffers and reagents. Nanospray interfaces especially benefit from low-volume, high-resolution separations. The mass spectrometer is a fast response system and benefits from separation speeds higher than normally supplied by HPLC systems. All of these requirements have provided constraints on new development directions for HPLC systems. 

Working CN standard solutions. Pipette 0, 1, 2, 5, 10, 20, and 40 mL of secondardy standard into 100-mL volumetric flasks and dilute first to 90 mL with caustic soda dilution solution and then to a final volume of 100 mL with distilled water after adding buffer and reagents. 

Pure water can be a major culprit, because it is the starting point for most eluents, standards, buffers, and reagents. Results are obviously meaningless if background contaminants in the water mask the very substances being analyzed. 

The system was equilibrated with a column temp, of 38 °C and flow rates of 20 mL/hr and 10 mL/hr for the buffer and reagent pumps, respectively. Temperature gradients changed at 15 "C/min. 

Buffer and reagent consumption, and hazardous waste generated from the use of radioactive probes and ethidium bromide stain, can be considerable. 

Use sterile Milli-Q water during the development of liposomal formulations. Sterilize by filtration. All buffers and reagents are stored at 4°C and are used within four weeks of preparation. 

A last method of localizing HRP is by oxidizing ionic silver to give a visible deposition of elemental silver as a label. This method uses buffers and reagents that require a low pH and cause destruction of cellular morphology. Therefore, this method is not recommended except for specific cases where discrete particulate label is needed. 

The lysis of cells on-chip (either after a cell-sorting step or fl-om mixed sample sources) will, undoubtedly, be an important step in the development of sample processing microfluidic devices for total sample handling of crude biofluids. There are many examples in the literature that describe the lysis of cells in microfluidic systems and that can be easily divided into two categories (1) those that utilize a chemical-based lysis and (2) those that employ a physical breakdown of the cell. Chemical cell lysis has been demonstrated using a variety of buffers and reagents and... 

Buffers and Reagents. All buffers can be made a day earlier and stored at 4° until ready for use, except that protease inhibitors should be added immediately prior to their use. 

Buffers and Reagents. Nucleation of clathrin lattices by API80 or AP2 was studied HKM buffer, consisting of 25 mM Hepes pH 7.4, 125 mM potassium acetate, 5 mM magnesium acetate, and 1 mM dithiothreitol. Lipid stocks and uranyl acetate stocks were prepared as above. 

Buffers and reagents for agarose gel electrophoresis (e.g., native 1-2% agarose gels in 0.5x TBE [see Appendix 3] containing 1 lig/ml ethidium bromide appropriate DNA size markers)... 

Buffers and reagents for SDS-PAGE (acrylamide/bisacrylamide, ammonium persulfate,... 

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