DOI: (Paper), 2016, 6, 101 Unusual surface and solution behaviour of keratin polypeptides Zhiming Lu a, Fang Pan a, Dong Wang b, Mario Campana a, Hai Xu b, Ian M. Tucker c, Jordan T. Petkov cd, John Webster e and Jian R.
As a manual positioning. Please also refer to the alternative Washburn-Method for powder contact angle with our Force Tensiometer – K100. Transmission rates. The needle NE50 with 5 µm internal diameter has been designed for the manual dosing of. Sensor of your tensiometer even when. Data transmission.
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Lu. a Biological Physics Laboratory, School of Physics and Astronomy, University of Manchester, Oxford Road, Manchester, M13 9PL, UK. E-mail: Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao 266580, China Unilever Research and Development Laboratory, Port Sunlight, Quarry Road East, Bebington, Wirral CH63 2JW, UK Menara KLK 1, Jalan Pju 7/6, Mutiara Damansara, 47810 Petaling Jaya, Selangor Darul Ehsan, Malaysia ISIS Neutron Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Science and Innovation, Campus, Didcot OX11 0QX, UK. Keratin production. White sheep wool was purchased from Brunswick Industrial Estate, Halifax, West Yorkshire, UK in the spring of 2011. Keratin K1S sample was obtained by following a recognised procedure with the application of some modifications.
The modification involved the immersion of 1 g of degreased wool (fine cuts) in 10 mL of the dissolving solution comprised of 8 M urea, 0.2 M SDS and 0.5 M Na 2S 2O 5. The mixture was then heated to 100 °C for a period of 30 min and filtered and washed through a stainless steel mesh. The filtered solution was then encased in cellulose tubing (molecular weight cut-off of 12 000–14 000 Da (Sigma)) at an ambient temperature of 18–20 °C for dialysis against distilled water.
The water was replaced every 4–5 hours during dialysis for approximately 4 days in order to remove excess reagents (urea, SDS and Na 2S 2O 5), with the final conductance close to that of the pure water. The obtained keratin stock solution was stored at 4 °C prior to subsequent use. Its concentration could be obtained by either UV absorption measurement or weighing the power after freeze drying a given amount of the stock. Investigation of the molecular weight distribution of the extracted keratin was carried out by SDS polyacrylamide gel electrophoresis (SDS-PAGE), using the Mini-PROTEAN 3 Cell system from Bio-Rad.
Molecular Dynamics
Stacking gels (6% acrylamide of about 0.75 mm thickness) and resolving gels (12% acrylamide of about 0.75 mm thickness) were prepared according to a standard method described by the Mini-PROTEAN 3 Cell Instruction Manual (run at a constant voltage of 150 V). Keratins were visualized by Coomassie Brilliant blue G 250 stain using a protein marker (Biolabs) for calibration. The molecular weight distribution of extracted wool keratins was observed as two main bands, at approximately 45 kDa (equivalent to type Ia keratins) and 60 kDa (equivalent to type IIa keratins). In addition, several weak bands were observed, corresponding to the low molecular weights at approximately 6–9 kDa and 10–20 kDa, which were attributed to the high-sulphur and high-glycine/tyrosine proteins of the matrix and the low-sulphur intermediate filament proteins. These parameters obtained from the wool keratin extractions are consistent with the results reported previously by other groups. For ease of data analysis in neutron reflection, it is necessary to use one keratin as a model molecule for the estimate of its scattering length density (SLD) and calculation of the amount of surface adsorption. Table SI1 in ESI shows how SLD values vary with the ratio of H 2O and D 2O due to the labile H/D exchanges.
An important observation is that the SLD values change little for different proteins in a given solvent such as H 2O. This illustrates that although proteins differ in sequence and in their physical and biological properties, all their amino acid compositions tend to be similar. Cryo-transmission electron microscopy. The cryo-TEM samples were prepared in a controlled environment vitrification system (CEVS).
∼4 μL sample solution was coated onto a TEM copper grid and the grid was blotted with two pieces of filter paper for about 2 seconds, leading to the formation of a solution thin film. Then, the grid was quickly plunged into a reservoir of liquid ethane (−165 °C, cooled by liquid nitrogen) and then kept in liquid nitrogen until the observation. After transferring the grid to a cryogenic sample holder (Gatan 626) and putting the holder into a JEOL JEM-1400 Plus TEM (120 kV) instrument at about −174 °C, one could observe the nanostructures. Crack for kitchendraw 6. Results (A) Surface tension Surface tension measurements were first carried out to outline the interfacial adsorption behaviour of the keratin solutions focusing on revealing their dynamic adsorption with time. These surface tension studies also provide an estimate of the concentration range required in the subsequent neutron reflectivity work. Shows the time dependent surface tension changes for a series of solutions prepared at pH 6. At the lowest concentration of 3 × 10 −4 mg ml −1, no surface tension reduction was observed over the entire measurement period.
As the keratin concentration was increased to 1 × 10 −3 mg ml −1, there was an initial induction period of some 3000 s, after which the surface tension showed a clear trend of decline. With further increase in keratin concentration, this induction period was seen to reduce, with the interfacial adsorption accelerating. As the keratin concentration was increased up to 0.1 mg ml −1, there was a further reduction of the induction time. Further increase in keratin concentration above that point was seen to produce relatively small changes in the surface tension profile, indicating the saturation of interfacial adsorption. On the basis of these and other surface tension measurements (not presented here), a concentration of 0.1 mg ml −1 was taken to be the approximate concentration around which the saturated keratin adsorption could be reached under these experimental conditions.
(B1) Reflectivity profiles of keratin in NRW subphase. Neutron reflectivity was initially used to determine the adsorbed amount of keratin at the air/water interface as a function of increasing protein concentration. This is usually carried out using a mixture of approximately 8% D 2O in H 2O, termed null-reflecting water (NRW).
NRW has the same scattering length density as air, and hence all reflectivity signal at this interface originates from the adsorbed interfacial layer. The measurements under this contrast can provide invaluable information regarding both adsorbed amount and interfacial thickness without any interference from water across the interface. Reflectivity profiles for a series of keratin solutions ranging from 3 × 10 −3 to 0.3 mg ml −1 at 25 °C, pH 6 and 5 mM NaCl are shown in, where reflectivity R is plotted against wave vector Q. The reflectivity can be seen to increase with increasing keratin concentration, and then to reach almost full adsorption at a concentration of 0.1 mg ml −1, followed by small changes when the concentration was further increased to 0.3 mg ml −1. It should be noted that time dependent effects were avoided in these NR measurements by ensuring that after the solutions had been loaded into the liquid troughs, a state of equilibrium was reached, with the timescales referred to from the surface tension data shown in.