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Canada Talkings

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Thomas Kopylov
Thomas Kopylov

Kosa - Detergent

We recommend a cold wash on a gentle cycle using natural detergent and hang drying for all our products. A colder wash saves electricity, the gentle cycle wears the fabric down less, natural detergent (especially with neutral PH) can retain the colour and elastane for longer and hang drying is easier on the clothing and the environment. Hand washing is always a good option too. If you are not separating delicates, use a laundry bag to protect the fabric from getting damaged. Look for a dense fabric for the laundry bag.

Kosa - Detergent

Tip: Spot treat any stains with cool water and a diluted solution of detergent. Test the solution on a part of the garment that's hidden or concealed, and if there is no adverse reaction, blot the stain with the solution before soaking. Be gentle when spot treating silk, as rubbing one spot too much can lighten the fabric.

Overall strategy. See text for details. (1) Cut-off: Proteins whose soluble fraction is less than 30% are subject to a detergent screen as illustrated for the human nucleotide excision repair protein (NER) XPD expressed in a baculovirus-infected Sf9 system. (2) Detergent extraction candidates: recombinant protein is extracted from insoluble pellets using a panel of detergents and the efficiency of each detergent to solubilize sample is quantified using immuno dot blots. (3) Small scale purification allows us to fine-tune salt and detergent concentration for sample binding to affinity beads and tag removal. (4) Large-scale purification with best detergent candidate. (5) Dynamic Light Scattering (DLS) and Multi Angle Light Scattering (MALS) experiments to test monodispersity. Aggregated samples (defined here as proteins whose measured radius (by DLS) is three times larger that it's predicted one) will be subject to additional detergent screening. (6) Detergent optimization: purified samples with large particle size are rescreened and analyzed in batch form using DLS. Detergents that can successfully produce monodisperse samples (measured radius

Biological activity of detergent-solubilized samples. (a) GαSβγ GTPγS binding activity comparison in select detergents. GTPγS binding of 800 nM GαSβγ was measured in the presence of 0.5 mM Brij-58 alone (red trace), 0.25 mM C12E9 (green trace) and 4 mM Fos-Choline-12 (blue trace) over 20 min. Each sample was passed through nitrocellulose membranes and GTPγS binding was quantified by measuring [35S]GTPγS-GαSβγ scintillation. (b) HepG2 cells cultured in the presence of Wnt-5A solubilized in Brij-58 but not DDMAB for 48 h led to a noteworthy decrease in TOPFLASH reporter activity. (c) Binding of human XPG to the Y shape DNA substrate. Increasing amounts of human XPG was added to 25 nM fluorescently labeled DNA substrate. The equilibrium dissociation constant, Kd, was estimated as the amount of XPG that was necessary to get to 50% maximum binding. (d) Binding of human XPD to a single strand 39mer DNA substrate. Increasing amounts of human XPD were added to 25 nM fluorescently-labeled DNA substrate. The equilibrium dissociation constant, Kd, was estimated as 58 nM using non-linear fit of the anisotropy to the XPD concentration.

Effect of detergent solubilization on particle radius for S. cerevisiae core TFIIH complex (cTFIIH, comprises Rad3, Tfb1, Tfb2, Ssl1 and Tfb4 [25]), human XPA, and human GαSβγ. The predicted radii for cTFIIH is 9.1 nm, human XPA is 2.5 nm and GαSβγ is 6.6 nm. Proteins whose purifications were initially successful but had large particle sizes were subject to DLS experiments in (batch mode) to find conditions that lead to monodisperse samples. Correlograms measure the exponential time decay of the autocorrelation function of the scattered light of particles in solution diffusing with Brownian motion and they contain information on the diffusion speed of different particle size groups in solution. After standard data analysis, from correlograms, shown in black in the top panel it is possible to estimate the radii of diffusing particles in solution. Larger decay times correspond to larger particle radii. Control correlograms are shown in top panels. Two examples of particle radius distributions are shown in lower panels. Indicated in blue are examples of detergent conditions with still large aggregates in solution. Examples of detergent conditions that had a dramatic effect on the particle radius are indicated in red. 041b061a72


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