Analysis Of Proteins Using Nearuv Spectrophotometry

This procedure identifies proteins using near-UV spectrophotometry. Protein solution is placed in a UV-transparent quartz cuvette, absorption of light is measured as a function of wavelength, and the resulting spectrum is transferred either to a displaying device (e.g., CRT or printer) for inspection or to a computer that can employ a spreadsheet or other program for derivative calculations and or quantitative analyses (see Support Protocol). Acid ethanol cleaning solution (see recipe) 4...

Selective Precipitation By Isoionic Precipitation Dialysis Method

One of the older means of rendering proteins salt-free or nearly salt-free (i.e., isoionic) is dialysis (also see unit 4.4 & appendix 3b). However, two problems frequently arise with conventional dialysis (1) when appreciable amounts of protein are present, osmotic effects result in swelling of dialysis bags as salt diffuses outward and (2) often it is quite uncertain where the isoionic point is, even if it is feasible to deionize by dialysis against a buffer. The resin deionization method...

Growth Of Mammalian Cells For Protein Expression

Mammalian cell culture media are complex in comparison to the simple defined media used for propagation of bacteria and yeast. This complexity stems from the need to satisfy the many functions required for normal growth and metabolism of mammalian cells (Bettger and McKeehan, 1986). Nutrients must satisfy the requirements for growth, maintenance of the cell, and expression of products. Additional specific growth factors such as insulin or insulin-like growth factor (IGF) are usually required....

Gel Dialysis Of Protein Samples

This protocol describes a simple method for dialyzing small interfering molecules and salts away from protein samples using an agarose or polyacrylamide gel (see Fig. 3.4.5 Freifelder and Better, 1982). Somewhat dense, well cross-linked gels are used to keep proteins from penetrating the gel while unwanted salts and low-molecular-weight compounds diffuse (are dialyzed away) into the gel. This is a micro to semimicro method, an alternative to conventional dialysis (Craig, 1967), and to the use...

Background Information

Under nondenaturing conditions, in which protein activity, native charge, and conformation are sustained, electrophoretic separation depends on many factors, including size, shape, and charge. Characteristics such as intrinsic molecular weight (i.e., in the absence of denatu-ration), the number of isoforms, and the presence of multimeric proteins can be determined with nondenaturing electrophoresis (often called native electrophoresis). The most important application of nondenaturing...

Info

Soluble in water (to 5 mM) and DMSO (to 50 mM) Soluble in water (to 5 mM) and DMSO (to 50 mM) aSee SUPPLIERS APPENDIX for addresses and phone numbers of manufacturers. -1 mg ml purified protein Coupling buffer (see recipe), ice cold 2 mg ml NHS-LC-biotin (Pierce, Vector Labs) in N,N-dimethylformamide (DMF), room temperature, prepared just before use 50 mM glycine in coupling buffer 0.22- m filter e.g., Spin-X centrifuge filter (Costar) or syringe filter (Nalgene) Additional reagents and...

Strategies For Isolation Of Insoluble Proteins

Recombinant proteins expressed in E. coli that are located in the low-speed pellet fraction (see Fig. 6.1.1) following cell lysis are highly aggregated (i.e., inclusion bodies). Inclusion bodies are normally derived from protein aggregation in the cytoplasm, or in the periplasm if a secretion vector was used. As mentioned above, protein can also be located in either the low- or high-speed pellet fractions because of interaction with bacterial nucleic acids. Furthermore, if the protein is known...

Desalting The Sample After Cysteine Modification

Except for the in situ method Basic Protocol 9 , all of the protocols presented leave the protein in the presence of high salt and reaction by-products which are usually not compatible with subsequent analytical techniques. This problem may be remedied by one of the following desalting methods. The methods are presented in increasing order of technological complexity. Protein recovery is a concern because small quantities of protein may be lost in dialysis or gel filtration. Modern...

Protease Cleavage Of Fusion Protein In Solution To Remove Gst Affinity

Uncleaved Thrombin Cleaved Protein

The glutathione-S-transferase GST affinity tag is removed by cleaving with thrombin pGEX-T vectors or factor Xa pGEX-X vectors . Conditions for optimal cleavage of each recombinant fusion protein must be empirically determined. Some of the parameters that can be varied include temperature, enzyme-to-substrate ratio, length of incubation, and buffer conditions. Proteolysis can usually be performed in the glutathione buffer used to elute the fusion protein from the column, either with or without...

Phenylthiocarbamyl Amino Acid Analysis

Phenylthiocarbamyl amino acid analysis PTC-AAA is an established method for picomole-level quantitative analysis based on the reaction of phenylisothiocyanate PITC with amino groups to form phenylthiocarbamyl amino acid derivatives, which are then separated by reversed-phase HPLC and identified and quantified by ultraviolet absor-bance. Since its introduction in the 1980s Koop et al., 1982 Heinrikson and Meredith, 1984 Tarr, 1986 , PTC-AAA has become the most popular precolumn-derivatization...

Graphical Determination Of Specific Productivity Of Cells Producing Heterologous Protein

For definition of specific productivity, see Support Protocol 6. Materials Spreadsheet software with graphical output e.g., Microsoft Excel or Lotus 123 1. Sample cultures at time points 1 and 2, count viable cells, and determine protein concentrations see Support Protocol 6, steps 1 and 2 . 2. Plot the viable cell densities versus time and plot the protein concentrations versus time on x-y formatted charts using spreadsheet software capable of graphical output. 3. Curve-fit the charts with the...

Measurement Of Dynamic Column Capacity And Breakthrough Capacity Of Ionexchange Columns

For chromatographic separations that will be performed on a routine basis as well as separations that will be scaled up, it is necessary first to optimize conditions with respect to resolution and to determine the capacity of the medium with respect to the target protein see Support Protocol 1 . Knowledge of the capacity allows optimal use of the gel medium in terms of cost and yield, and also allows working limits to be set on the sample load to ensure robustness of a particular purification...

Peptide Sample Preparation by Ether Precipitation

This procedure is suitable for precipitating peptides from organic acids and is used routinely for concentrating synthetic peptides from the trifluoroacetic acid cleavage cocktail following 9-fluorenylmethyloxycarbonyl Fmoc solid-phase peptide synthesis. It can be used to desalt peptides in nonvolatile buffers but is not recommended for removing detergents like SDS. Take appropriate safety precautions when using this technique ether is highly flammable, and a refrigerated centrifuge and fume...

Differential Scanning Calorimetry

Before beginning calorimetry, an instrument baseline is established by a buffer buffer scan. If there are changes in the shape and or relative position of the buffer buffer scan, the cells are probably dirty see Support Protocol 4 for cleaning procedure . Protein solution, dialyzed unit4.4 amp appendix3.b and in dialysis bag see Critical Parameters Equilibration buffer buffer solution used for the last dialysis see Critical Parameters for discussion of buffer selection Spectrophotometer and...

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Figure 6.1.2 Localization of secreted and periplasmic proteins in E. coli. Periplasmic protein produced via a secretion vector can leak into the medium and be recovered by centrifugation supernatant, S1 or filtration. Washing cells with an isotonic solution such as lightly buffered 0.15 M NaCl or 0.25 M sucrose can also release protein S2 . The compartmentalized periplasmic proteins are released by isotonic shock treatment by directly suspending normal cell paste or plasmolyzed cell paste into...

Reagents And Solutions

Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock solutions, see APPENDIX 2E for suppliers, see SUPPLIERS APPENDIX. 100 mM Tris - Cl, pH 7.8 pH as determined at 4 C 100 mM Tris - Cl, pH 7.8 pH as determined at 4 C 10 mM EDTA from 0.5 M stock 0.5 mM phenylmethylsulfonyl fluoride PMSF 5 mM benzamidine - HCl 780 mg liter Prepare immediately before use Purification of PMSF can be added as 2.5 ml liter of 0.2 M PMSF stock solution. 60 mM ethanolamineHCl, pH...

Osmotic Dehydration

Although ultrafiltration is a highly effective procedure for concentrating relatively large volumes, it may be less effective and convenient with small volumes and high protein concentrations. Osmotic dialysis relies on the use of a high concentration of material outside a dialysis tube to draw water and small-molecule solutes from its contents by osmosis, leaving a more concentrated protein solution within the dialysis tubing. Either sucrose or polyethylene glycol PEG Mr 15,000 to 20,000 may...

Diafiltration Or Concentration By Tangentialflow Ultrafiltration

The most rapid method for dialyzing large samples is the use of ultrafiltration membranes fabricated into hollow fibers or spiral-wound layers, or, as in this protocol, in the form of stacked plates. The sample is allowed to flow tangentially over these membranes with recirculation. The semipermeable nature of the membrane then permits water and materials with molecular weights that are substantially below the MWCO value to cross through the membrane surface relatively unimpeded retained...

Hartreelowry Assay For Quantitation Of Total Protein

The original Lowry method for total protein analysis was first described in one of the most cited papers in biochemistry Lowry et al., 1951 . The assay is a colorimetric assay based on cupric ions and Folin-Ciocalteau reagent for phenolic groups. The assay has been reinvestigated many times and sometimes improved. Most of these studies were designed to discern how interfering compounds distort the assay and how detergents solubilize otherwise insoluble proteins. The literature related to this...