Peptides solution

Fig. 12 Pn-4 (a, b) and Pn-8 (c, d) P-sheet variation measure by IR circles) and NMR triangles). 10 mg mL peptide solutions prepared in (a, c) D2O and (b, d) 130 mM NaCl in D2O. For Pii-4, I nematic gel, II flocculate, III nematic fluid, IV isotropic fluid. For Pn-8, I isotropic fluid, II biphasic solution. III nematic gel. Adapted from Carrick et al. [23]. Copyright 2007, with permission from Elsevier...

The 2 pi of the concentrated peptide solution was mixed with 4 pi of 2,5-dihydroxybenzoic acid [ 7 mg in 500 pi of the mixture of acetonitrile/0.1% trifluoroacetic acid, 1/2 (v/v)]. The resulting mixture was applied to the MALDI steel plate and left to crystallise. Afterwards, the MALDI-TOF mass spectra were measured by a BIFLEX IV instrument (Bruker, Germany) under appropriate conditions (potential 19 kV on the plate and 15.05 kV on the deflector, positive reflector mode potential 20 kV, laser intensity 50 or 60%). The range of detected masses was from 600 to 3000 Da. For the external calibration the standard mixture of peptides M-Pep was used. 

Add 10 pi of the SATP solution to each ml of protein or peptide solution. 

Apply to the column 1.0ml of peptide solution (dissolved in equilibration buffer) to be reduced. Normally, small peptides (molecular weight less than or equal to that of insulin) require no deforming agent (denaturant) such as guanidine to be completely reduced. 

Add sodium borohydride (Aldrich) to the peptide solution to obtain a final concentration of 0.1M. Generation of hydrogen bubbles will occur as the borohydride is dissolved. 

Quench the oxidation by the addition to the peptide solution of at least a 4-fold molar excess of N-acetylmethionine or sodium sulfite over the concentration of periodate in the reaction mixture. Pre-dissolve the quencher in buffer at a higher concentration prior to adding an aliquot of it to the reaction solution. React for 10 minutes. 

Add 1.5 pi of the o-methylisourea hemisulfate solution to the peptide solution with mixing. 

Add the solution prepared in step 2 to the protein solution to obtain at least a 10-fold molar excess of small molecule to protein. In the case of the peptide-protein immunogen conjugate, add the 500 pi of peptide solution to the 200 pi of protein solution. 

Add 500 pi of the peptide solution to 200 pi of carrier protein. For greater reaction volumes, keep the molar ratio of peptide-to-carrier addition the same and proportionally scale up the amount of EDC added in the next step. If the peptide is initially dissolved in DMSO, much less peptide volume compared to protein volume should be used to maintain solubility (see discussion in step 2). 

Add 10 pi of the peptide solution before conjugation to the appropriate wells in duplicate. 

Mix 0.5 ml of the peptide solution with 0.5 ml of the carrier protein solution. Chill on ice. Add 0.4 ml of the fois-diazotized tolidine solution. There should be a color change from orange to red almost immediately. Continue the reaction for 2 hours on ice in the dark. 

Dissolve a sulfhydryl-containing peptide hapten at a concentration of 25 pmol/ml in degassed, nitrogen-purged lOmM HEPES, 0.15M NaCl, pH 7.0. Add the peptide solution to the liposome suspension at a molar ratio necessary to obtain at least a 5 1 excess of thiol groups to the amount of maleimide groups present (as MPB-DPPE). 

Figure 5.25 AFM images of intermediate stmctures in self-assembly of peptide KFE8 in aqueous solution deposited on freshly cleaved mica surface (a) 8 min after preparation of solution. Inset electron micrograph of sample of peptide solution obtained using quick-freeze deep-etch technique (b) 35 min, (c) 2 h, and (d) 30 h after preparation. Reprinted with permission from Ref. 110. Copyright 2002 by the American Chemical Society.

A/1 it was possible to follow the growth of twisted ribbons, with a periodic twist of 80-130 nm, by depositing seeds on mica prior to the injection of a fresh peptide solution (Fig. 4C Goldsbury et al., 2005). In the case of human amylin, it was even possible to observe by time-lapse SFM how fibrils are formed from an oligomeric nucleus by initial growth in height from... 

The sample can be prepared in a cosolvent, such as 5-50% acetic add in water, acetonitrile/ water, chloroform, methanol, and trifluoroacetic acid (0.1 to 25%), at a concentration of approximately 1 to 5 mg mL-1. Sample solubility in this cosolvent is crucial because it acts as a medium between the sample and the matrix. The peptide solution (1 1 pL) is dissolved directly into the matrix on the probe tip (a flat round surface approximately 2-5 mm in diameter). When large amounts of sample (hundreds of micrograms) are available, the peptide can be added directly into the matrix, followed by the addition of a cosolvent such as trifluoroacetic acid (TFA) which, in addition to sample matrix solubility, can also facilitate protonation. 

The function of acidic peptides was examined by using the peptide which was isolated from beef soup and sequenced as Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala by Yamasaki and Maekawa (5) and Tamura et al (6). According them, the peptide solution tasted just like beef soup. Spanier (7) nam this peptide as Beefy Meaty Peptide (BMP) based on its taste. 

Peptide solution/suspension at 1 mg/mL in phosphate-buffered saline (PBS) stored at-20°C... 

Table I. G Values0 X 100 Observed at Irradiation Doses up to 5.0 Megarads in 2% Amino Acid or Peptide Solutions Saturated with Helium (02 free) or Oxygen at Three Temperatures...

Table III. Effect of Irradiation Temperature on Yields of Irradiation Products in 2% Amino Acid and Peptide Solutions...

Dissolve up to 4 mg of the peptide or hapten to be coupled in 1 ml of the reaction buffer chosen in step 1. If the peptide to be coupled is already in solution, it may be used directly if it is in a buffer containing no other amines or carboxylic acids and is at a pH between 4.7 and 7.2. Note If an assessment of the degree of peptide coupling is desired, measure the absorbance at 280 nm of the 1 ml peptide solution before proceeding to step 3. In some cases, a dilution of the peptide solution may be necessary to keep the absorbance on scale for the spectrophotometer. If the peptide is sparingly soluble in aqueous solution, it may be dissolved in DMSO and an aliquot added to the carrier solution. See the previous discussion on carrier proteins to determine the levels of DMSO compatible with carrier protein solubility. 

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