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HiTrap™ Desalting Columns with Sephadex G-25 Resin

Supplier: Cytiva

Description: HiTrap Desalting are ready-to-use 5 ml columns prepacked with Sephadex G-25 Superfine resin for fast and convenient desalting and buffer exchange.

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N-Octyl-β-D-glucopyranoside non-ionic detergent, Pierce™

Catalog Number: (PI28310)

Supplier: Invitrogen

Description: Thermo Scientific Octyl-beta-Glucoside is a nonionic detergent that has been widely used for membrane protein solubilization.

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Anti-ADH6 Rabbit Polyclonal Antibody

Catalog Number: (76075-912)

Supplier: Prosci

Description: For WB starting dilution is: 1:1000 For IHC-P starting dilution is: 1:50~100 For FACS starting dilution is: 1:10~50

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AASTY 6-45

Supplier: CUBE BIOTECH

Description: AASTYs (Acrylic acid-co-styrenes) - like AASTY 6-45 - are highly alternating copolymers, well-suited for generating native lipid nanodiscs. They are a 2022 novel developed series for membrane protein solubilization & stabilization. AASTY 6-45 gets it's name from its molecular weight and Acrylic Acid : Styrene Ratio. These varying ratios of acrylic acid to styrene contribute to the hydrophilic properties of our AASTYs. In general lighter AASTYs, like 6-45 tend to be more aggressive, while heavier AASTYs, such as 11-45 show higher thermodynamic stability.

The exact composition of AASTY copolymers shows different extraction efficiency, depending on the lipid composition of the lipid bilayers being formulated into nanodiscs. As AASTY is made by controlled radical polymerization techniques, the dispersity of polymer molecular weight distribution is low, and the molecular weights are controlled. This means that excess AASTY copolymer can be removed by dialysis after nanodisc formation. Based on previous findings on SMA, it is the expectation that AASTY of different molecular weights will display different rates of nanodisc formation, extraction efficacy, and stability of resulting nanodiscs.

Every membrane protein solubilization needs to undergo a screening process before. The characteristic phospholipid environment surrounding the different membrane proteins in question performs differently well with each polymer. To support you in this process we offer a handy Screening Kit for AASTYs to test them all. Additionally, we recommend the two following publications if you would like to get further information: Smith et al. 2020 & Timcenko et al. 2022

Urea ≥99%, white prills, Ultrapure

Supplier: MP Biomedicals

Description: Urea is the principal end product of nitrogen metabolism in most mammals, formed by the enzymatic reactions of the Kreb's cycle.
Urea is a mild agent usually used in the solubilization and denaturation of proteins. It is also useful for renaturing proteins from samples already denatured with 6 M guanidine hydrochloride such as inclusion bodies; and in the extraction of the mitochondrial complex. It is commonly used to solubilize and denature proteins for denaturing isoelectric focusing and two-dimensional electrophoresis and in acetic acid-urea PAGE gels. Urea is used in cell or tissue culture media to increase the osmolality. Urea has also been used as fertilizer because of the easy availability of nitrogen; in animal feeds; it is reacted with aldehydes to make resins and plastics; condensed with malonic ester to form barbituric acid; used in the paper industry to soften cellulose; used as a diuretic; enhances the action of sulfonamides; an antiseptic.
Urea in solution is in equilibrium with ammonium cyanate. The form that reacts with protein amino groups is isocyanic acid. Urea in the presence of heat and protein leads to carbamylation of the proteins. Carbamylation by isocyanic acid interferes with protein characterization because isocyanic acid reacts with the amino terminus of proteins, preventing N-terminal sequencing. Isocyanic acid also reacts with side chains of lysine and arginine residues resulting in a protein that is unsuitable for many enzymatic digests. In addition, carbamylation often leads to confusing results from peptides having unexpected retention times and masses. When performing enzymatic protein digests it is important to remove urea first. Even though some enzymes will tolerate small amounts of urea, the elevated temperature used for most reactions will lead to carbamylation during the course of the digest. The urea can be removed prior to digestion by fast reversed phase chromatography, spin columns, or dialysis.
Dissolve urea in deionized water to the desired concentration.For every 10 ml of solution, add 1 g of Amberlite® IRA-910.Stir for one hour at room temperature

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