Protein optimization

Name Density (g/mL) % Lipid % Protein Optimal (mg/dL) Poor (mg/dL)... 

An alternative protocol for labeling sulfhydryl-containing proteins that does not require DTT reduction can be found in a method adapted from Ando (1984). When preparing any fluorescently labeled protein, optimization of the dye-to-protein ratio is important to obtain the best performance in the intended application. 

Each bead can iodinate up to 500 pg of tyrosine-containing protein or peptide. This translates into an oxidative capacity of about 0.55 pmol per bead. The rate of reaction can be controlled by changing the number of beads that are used and altering the sodium iodide concentration added to the reaction. Reaction volumes of 100-1,000 pi are possible per bead. The following protocol is suggested for iodinating proteins. Optimization should be done to determine the best incorporation level to obtain good radiolabel incorporation with retention of protein activity. 

Fig. 8. SB 242084 is a agonist for PLC-PI and an inverse agonist for PLA2-AA and GTP[y35S] binding to Gf/I (i.e., SB 242084 is a protean ligand) for the 5-HT2C receptor. Accumulation of IP and release of AA were measured in CHO-1C7 cells, which express a high level of the human 5-HT2C receptor (10-20 pmol/mg protein, optimized to detect...

INTERNAL PROTEIN SEQUENCING OF SDS-PAGE-SEPARATED PROTEINS OPTIMIZATION OF AN IN GEL DIGEST PROTOCOL... 

The differences in binding orders between TBPA and TBG suggest that different structural features may play a key role in receptor interactions. It has been shown (4,28) that TBG also preferentially binds to a tetraiodo-4 -phenoxide ion, but since Ti is the strongest binder, this suggests a different side chain stereochemistry. Here we can assume that it is the twist-skewed diphenyl ether conformation which orients the Tside chain for optimal receptor-hormone interactions. In the case of the nuclear proteins optimal binding is observed for a distally oriented 3 -I and a 4 -hydroxyl. Side chain requirements appear to be similar to those of TBG (28,31). 

A general outline of an inclusion body protein recovery/purification scheme is present in Figure 2. For a given protein, optimal conditions for each individual step have to be established and are a function of the composition of the starting material and the characteristics of the protein including protein size, the presence of inter and/or intramolecular disulfide bonds, the number and types of subunits in the protein molecule, and the presence of prosthetic groups. 

Inactivation of phage T2 in p-lactamase solution by QAC-treated beads is shown in Figure 4. The initial T2 titer was 3.0 x 10 PFU/ml. A quantity of 0.8 g of beads were mixed with 10 ml of p-lactamase solution. Fifteen percent of the viruses survived this treatment. The amount of total protein in the solution was 80% of the initial value after the adsorption process, while the recovery of p-lactamase activity was at least 70%. It was the purpose of this experiment to demonstrate that QAC-treated beads can effectively remove viruses from a protein solution without significantly losing the activity of the protein. Optimal adsorption condition and mode of operation ought to be determined by studying the interactive effects of pH, ionic strength, and temperature of the solution, with the specific t)qjes of virus and protein of interest. 

Fig. 6.2 Schematic overview of the linear-scaling ONIOM approach chosen for protein optimization...

Compared to other techniques used for protein optimization, the WalkThrough recombination technique presented here shows several advantages for in-vitro protein optimization ... 

The electroporation parameters used in this protocol are suitable for transferring antibodies to the cytoplasm but may not be for incorporating antibodies into the nucleus for studies on the functions of nuclear proteins. Optimization of the electrical parameters is essential for such application. 

If the given rapid dilution protocol does not work out, reduce the final protein concentration (e.g., double refolding buffer to 200 volumes). Make sure your unfolded protein is pure (>90 %). Alternatively, refolding by dialysis can be tried [30, 31], however, we obtained our best result with the described rapid dilution procedure. If the protocol is transferred to other proteins, optimization of refolding work-up might be necessary. We recommend screening various concentrations and ratios of salts, supplements, pH values, and redox systems in analytical scale, similar as described in [37]. 

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