Buffers urea containing

For precipitated protein, buffered solutions containing chaotropic reagents such as 0.1% SDS, 8 M urea, or 6 M guanidine or proteolytic enzymes such as pepsin may be used. However, an extended washing with buffer is required to remove SDS and guanidine. Unexpected elution behavior can occur if these reagents are not removed completely. 

The caseins may be quantified by densitometrically scanning polyacrylamide gel electrophoretograms (section 4.4.1) but more quantitative results are obtained by ion-exchange chromatography using urea-containing buffers. However, it should be realized that the specific absorbance of the individual caseins differs greatly (Table 4.2). 

A buffer solution containing urea flows along one side of a flat membrane and the same buffer solution without urea flows along the other side of the membrane, at an equal flow rate. At different flow rates the overall mass transfer coefficients were obtained as shown in Table P8.1. When the liquid film mass transfer coefficients of both sides increase by one-third power ofthe averaged flow rate, estimate the diffusive membrane permeability. 

Fig. 3. Time course of S. cerevisiae ure2dal80 mutant strain culture in pH 6.5 buffered medium containing proline, urea, or ammonium sulfate as nitrogen source.

Proteins were then dissolved in 20 mL of 0.125 M Tris-HCl, pH 7.4 containing 1% (m/v) CHAPS and the protease inhibitor cocktail, and treated with CPLL. With several variations from the standard treatments such as other complementary extractions from seeds, the use of urea-containing extraction buffer, and an additional peptide ligand library, the total number of detected gene products was quite large. Two hundred and thirty one unique proteins were found in the pulp (vs. 56 described previously) and 61 in the seeds (vs. only four reported by the literature). In the latter case, the presence of seed storage proteins, oleosins, and histones were detected in the pulp, a thaumatin-like protein, an allergenic protein also named Ole el3, was confirmed. 

For the preparation of gel-filled capillaries, a solution consisting of 50 mM tris, 50 mM boric acid, and 7 M urea (pH - 8.3) was used both to prepare the gel and as the running buffer. For some of the gel-filled capillaries used in this work, the buffer also contained 3% PEG 20,000 Q5) (Fluka Chemical Corp., Ronkonkoma, NY). The fused-silica capillaries were rinsed with 1 N HC1, IN NaOH, and then methanol. A 1 1 mixture of... 

Gilar et al. have also applied CE to the separation of dihydropyridine calcium antagonists [25]. Their method used an uncoated capillary (76 cm x 75 pm i.d.) at 20 kV, a running buffer of 20-25 M phosphate-borate buffer (pH 9-9.5) containing 1% urea, and detection at 240 nm. The buffer also contained 10 mM p-cyclodextrin, and varying amounts of SDS. 

Figure 7 Electrophoretic separation of DNA sequencing fragments generated on ssM13mp18 with BigDye-labeled universal (-21) primer and AmpliTaq FS at the optimum experimental conditions 2.0% (w/w) LPA 9 MDa and 0.5% (w/w) 50 kDa LPA, 200 V/cm, and 60°C. Conditions effective length C = 30 cm (L = 45 cm), 75pm i.d., 365 pm o.d. polyvinyl alcohol-coated capillary running buffer (both cathode and anode), 50 mM Tris/50 mM TAPS/2 mM EDTA. Cathode running buffer also contained 7 M urea, the same as in the separation matrix. The samples were injected at a constant electric field of 25 V/cm (0.7 pA) for 10 s and electro-phoresed at 200 V/cm (10.2 pA) at 60°C. (Reproduced with permission from Ref. 88.)...

Fig. 15. Absorption spectra of creatine phosphokinase in 8 M urea-0.1 M acetate buffer, pH 4.0 after treatment with different amounts of NBS. Numerals indicate moles of NBS per mole of protein. Samples were read aKainst a urea-buffer blank containing identical concentrations of NBS. From Quiocho et al. (1961).

Inner root sheath proteins have been obtained from tissue dissected from the roots of rat vibrissae and porcupine quills and then extracted with 8 M urea containing iodoacetate and pH 8 buffer. The inner root sheath proteins (Table VI) and those isolated from the medulla of hair or porcupine quill are unusual in containing citrulline in protein combination (Rogers, 1958, 1962 Allen et al, 1964). 

The reduced and carboxyamidomethylated LO was then lyophilized and redissolved in 100 mM NH4HCO3 (pH 8.0) containing 2 M urea at 37°C, with shaking. The digestion buffer also contained 5 mM CaCl2, which is an absolute requirement for thermolysin activity (21). Proteolytic digestion was initiated by the addition of thermolysin to 4% (w/w) of the total protein weight. A second... 

Figure 7.5 2D-PAGE of the wine proteins (cv. Incrocio Manzoni 6.0.13) (Polesani, 2004). Proteins from 800 p.L of dialyzed/freeze-dried wine dissolved in 100/xL of 25 mM Tris-HCl buffer pH 9.0 containing urea 8M (buffer A), then reduced with 5mM tributylphosphine (90min, room temperature), alkylated with iodoacetamide (20 mM, 90 min at room temperature) and diluted with 100/xL buffer A containing 3M thiourea and 8% CHAPS. An IPG strip (7cm, pH 4-7) was soaked in the sample (200/xL) for 12h. IEF performed at 1000V for lh and 4000V for 6h. Strip incubated in lOmL of 0.1 M Tris-HCl buffer pH 6.8 containing 2% SDS, 5% 2-mercaptoethanol and 20% glycerol for 25 min and loaded on a SDS-PAGE gel...

Staining solution for polyacrylamide gels run in alkaline or acidic buffers and for urea-containing gels... 

NB Solubilities were determined in Britton-Robinson buffers. Aldiou the solubilities increased in buffer solutions containing 2-5% w/v urea, the pXa value remained constant. See Glibenclamide for further details. 

Analysis of immunoprecipitated RNA. The supernatants of the immunoprecipitation reactions are collected for analysis and the beads washed four times for 5 min each with 1 ml of ice-cold IPPiso- The precipitated RNA is released from the beads by digestion of proteins at 37°C for 30 min in 200 p of proteinase K buffer (50 mM Tris-HCl, pH 7.6 10 mM EDTA, 1% (w/v) SDS, and 0.8 mg/ ml proteinase K). RNAs from both the supernatants and precipitates are isolated by phenol extraction and ethanol precipitation, and analyzed on 7 M urea-containing 8% polyacrylamide gels. A preprecipitation sample consisting of the same amount of RNA used in the reaction should be included to the gel to quantitate the results. 

Cells or subcellular fractions were harvested in SDS and urea containing lysis buffer exactly as described [5], and total cellular homogenates analyzed by one- and two-dimensional gel electrophoresis [7]. The gels were both stained with Coomassie brilliant blue-R and processed for fluorography as described [5]. 

Nuclei from 2 g tissue were prepared by the method of Utakoji [1], and incubated for 30 min at 20°C in 2 ml of 0.25 M buffered sucrose containing 10 mAf Tris/HQ pH 8.2, 10 roM MgCl2,5 mM 2-mercaptoethanol, 5 mM NaF, 50 mM NaQ, 0.5 mM PMSF and 2 juQ [ C]-NAD [1]. Nuclei were washed two times in cold buffer to remove free label and extracted three times in ice-cold PCA. After centrifuging, the pellet was reextracted with 0.25 N-HQ and soluble proteins in the PCA, and HCl supernatants analysed on 20% acid urea polyacrylamide gels at pH 2.9 [13]. Gels were either stained directly with 0.05% Coomassie Blue Rin methanohacetic acid.water (40 7 53 by vol) or cut into 5 mm slices which were then dissolved in 30% H2O2 at 50°C for 16 h followed by counting in 5 ml Packard scintillator 299. 

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