Phosphate buffer saline

Adsorption of t-PA to process equipment surfaces consisting of either stainless steel or glass was minimized by adding the detergent polyoxyethylene sorbitan monooleate (Tween 80) to the semm-free culture conditioned media at 0.01% (vol/vol). The equipment was also rinsed, before use, with phosphate buffered saline (PBS) containing 0.01% Tween 80. Hydrophilic, plastic equipment was used whenever possible. AH buffers were sterile filtered. Sterile filtration of Hquids and gases is usually carried out using 0.2 or 0.45 p.m filters. 

DI = deionized water PBS, phosphate-buffered saline HSA, human semm albumin essential medium + 10% fetal bovine semm. 

Calf kidneys, dog kidneys and rhesus monkey kidneys were treated with trypsin to give suspensions of cells. The suspensions were centrifuged and the packed cells diluted with 400 volumes (calf cells) or 200 volumes (dog cells and rhesus monkey cells) of a growth medium consisting of 5% horse serum and 0.5% lactalbumen hydrolysate in Earle s saline, with 100 units/ml each of penicillin and streptomycin. These media were used separately to produce Semliki Forest/calf interferon, Semliki Forest/dog interferon and Semliki Forest/rhesus monkey interferon. The cell-containing growth medium was dispensed into 500 ml medical flat bottles (70 ml in each). The cultures were incubated at 36°C. Confluent sheets of cells (monolayers) were formed in 5 to 6 days. The growth medium was then removed and the monolayers were washed with isotonic phosphate-buffered saline, pH 7.5. 

For Yiv > YPv> where y v and Ypv are the surface tensions of liquid and protein, respectively, AFads increases with increasing ysv, predicting decreasing polymer adsorption. An example of this is phosphate buffer saline where y]v = 72.9 mJ/m2 and Ypv is usually between 65 and 70mJ/m2 for most proteins [5]. Therefore, supports for gel-permeation and affinity chromatography should be as hydrophilic as possible in order to minimize undesirable adsorption effects. 

FIGURE 9.4 Circular dichroistn (CD) spectrum of dilute solution (0.24 mg/mL) of recombinant resilin in phosphate-buffered saline (PBS). (From Whelan, A.J. and Robinson, A.J., unpublished data). 

FIGURE 9.6 DSC of (a) recombinant resilin in water showing no enthalpic events, (b) bovine serum albumin in phosphate-buffered saline (PBS) showing denaturing occurring at 62°C, and (c) wool fiber in water showing denaturing of the a-helix at 145°C (Endotherm up). 

FIGURE 9.8 Typical stress-strain plots for a strip of recombinant resilin tested in phosphate-buffered saline (PBS). Sample cycled to 225%, showing resilience of 97% (solid curve) and later tested to failure showing extension at break of 313% (dotted curve). (FromElvin, C.M., Carr, A.G., Huson, M.G., Maxwell, J.M., Pearson, R.D., Vuocolo 1, T., Liyon, N.E., Wong, D.C.C., Merritt, D.J., and Dixon, N.E., Nature, 437, 999, 2005.)... 

FIGURE 9.9 Small dumbbell-shaped sample of recombinant resilin prepared with embedded fine stainless steel mesh tabs (left) and being tested in phosphate-buffered saline (PBS) (right). 

In a subsequent study, the effect of reducing the ELP molecular weight on the expression and purification of a fusion protein was investigated. Two ELPs, ELP [V-20] and ELP[VsA2G3-90], both with a transition temperature at 40°C in phosphate-buffered saline (PBS) containing 1 M NaCl, were applied for the purification of thioredoxin. Similar yields were observed for both fusion proteins, resulting in a higher thioredoxin yield for the ELP[V-20] fusion, since the ELP fraction was smaller. However, a more complex phase transition behavior was observed for this ELP and therefore a selection of an appropriate combination of salt concentration and solution temperature was required [39]. 

Figure 8. Standard curves for PbTx-2 ( ) and PbTx-3 ( ) in the brevetoxin radioimmunoassay. Lower detection limits are 0.2 — 0.5 ng in phosphate-buffered saline (PBS). Standard RIA conditions [ H]PbTx-3 = 1 nM antiserum dilution = 1 2000 sample vol. = 1 ml buffer = 0.1 M PBS, pH = 7.4.

PAS Periodic acid-SchiflF reagent PBA Polyclonal B cell activators PBC Primary biliary cirrhosis PBL Peripheral blood lymphocytes PBMC Peripheral blood mononuclear cells PBN N- e f-butyl-a-phenylnitrone PBS Phosphate-buffered saline PC Phosphatidylcholine... 

The specifications and standardization include raw materials, preparation method of the standard solution, concentration of proteins, and the main band on SDS-PAGE. The outline of the procedure for preparation of the calibrators is shovm in Eig. 4.2. Table 4.5 shows the raw materials and the preparation method of the initial extract. To prepare the calibrators, the raw materials are extracted by the standard solution containing SDS and mercaptoethanol. The initial extract is prepared by centrifugation and filtration of the extract. The diluted extract is then prepared by 10-fold dilution of the initial extract with phosphate-buffered saline (PBS pH 7.4). The protein concentration of the diluted extract is assayed using the 2-D Quant kit (Amersham Bio Sciences). The standard solution is then... 

Enzyme-linked immunosorbent assay (ELISA). Li and Li developed an ELISA procedure for imidacloprid to determine its residues in coffee cherry and bean extracts. A 25-g amount of sample extracted with 300 mL of methanol and 1% sulfuric acid (3 1, v/v) for 3 min. An aliquot of the sample extract (0.5 mL) is mixed with 1 mL of water and a gentle stream of nitrogen is used to evaporate methanol. The solution is then extracted with 1 mL of ethyl acetate, the extract is reconstituted in 1 mL of PBST (phosphate-buffered saline containing 0.05% Tween 20) and competitive ELISA is performed to quantify imidacloprid in the extract. Eor methanol extracts of coffee cherries and beans fortified with imidacloprid at 0.5 mgL recoveries of imidacloprid by the ELISA method were 108 and 94, respectively. 

Instruments, Inc., Rockville, MD) was added, and the proteins were fractionated using a 10-channel RIEF apparatus. The proteins in each of the 10 fractions were then dialyzed against large volumes of phosphate-buffered saline (PBS, pH 7.4). Immunoelectrophoresis and a radioimmunoassay (RIA) for PCP were used to determine the RIEF fractions with the greatest purity and anti-PCP activity. 

The disassembly rate of dendritic molecules 18 and 19 was evaluated in phosphate buffered saline (PBS, pH 7.4) in the presence and absence of PGA. The release of tryptophan was monitored by a reverse-phase HPLC at a wavelength of 320 nm. The results are presented in Figs. 5.11 and 5.12. No disassembly of either system was observed in the buffer without PGA (data not shown). In the presence of PGA, dendritic molecule 18 disassembled to release tryptophan within approximately 4 days (Fig. 5.11), whereas dendritic molecule 19 released its tryptophan tail units within 40 min (Fig. 5.12). 

Dendron 32 was incubated in phosphate buffer saline, pH 7.4, in the presence and in the absence of PGA. The progress of the disassembly was monitored by RP-HPLC, and the results are presented in Fig. 5.27. Tryptophan was gradually released from dendron 32 upon incubation with PGA. The release was completed within 48 h in the presence of PGA the control reaction without PGA showed no release at all. Although the disassembly of this dendron occurred more slowly under physiological conditions than dendron 31 in the MeOH/DMSO environment (Fig. 5.24), PGA cleaved its phenylacetamide substrate from dendron 32 and the resulting amine intermediate was disassembled to release the total six molecules of tryptophan. 

Preparations of PEG-modified proteins. A. SC-PEG (1 g, 0.2 mmol) was added to a stirred solution of Bovine Serum Albumin (BSA) (100 mg, 1.5 x 10 6 mol) in 0.1 M sodium phosphate, pH 7.8 (60 mL). Sodium hydroxide (0.5 N) was used to maintain pH 7.8 for 30 min. The excess of free PEG was removed by diafiltration using 50 mM phosphate buffered saline. Approximately 30 amino groups of the native protein were modified as determined by trinitrobenzenesulfonate (TNBS) assay (28). The same degree of modification was obtained when the experiment was repeated under identical conditions using SS-PEG instead of SC-PEG. 

Concentration and MWD of F-PHEA After Absorption. F-PHEA was determined in perfusate samples by quantitative GPC relative to a freshly prepared F-PHEA standard run on the same day. Either a mixed-bed column (12 x 300 mm Sephacryl S-200 Sephadex G-25 SF 3 1, Pharmacia LKB) or a Separon HEMA-Bio 40 column (8 x 250 mm 10 pm particle size, Tessek A/S, Aarhus, Denmark) was used with a 20 pL injection volume. A mobile phase of pH 7.4 phosphate buffered saline (0.05 M phosphate, 0.15 M NaCl) was supplied (Model LC-7A Bio Liquid Chromatograph, Shimadzu Corporation, Kyoto, Japan) at 0.5 or 1 mL/min. Fluorescent detection was employed (Model RF-535 Fluorescence HPLC Monitor,... 

Figure 16 Pulsatile release rate of indomethacin in response to a stepwise temperature change between 20°C and 30°C in phosphate-buffered saline (pH 7.4). (From Ref. 36.)...

Prothymosin ct is a highly acidic protein of 109 residues that is unfolded at neutral pH, as demonstrated by CD and small-angle X-ray scattering (Gast et al., 1995). Figure 33 shows the CD spectra of prothymosin a under several conditions. In phosphate-buffered saline at pH 7.4 and 20°C, it has —1000 deg cm2/dmol, corresponding to 30%... 

Place 3 ml of sterile 0.5% phosphate-buffered saline on a charcoal agar slant. 

Figure 8 Observed longitudinal (/ 0h, circles) and transverse (r2obs, squares) relaxivity for 0.1 mM MS-325 in the presence (filled symbols) and in the absence (open symbols) of 22.5% (w/v) HSA at 37 °C, phosphate-buffered saline, pH 7.4 (reproduced by permission of the American Chemical Society from J. Am. Chem.

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