Freezing protein alterations from

The risk of chemical instability can be assessed from the primary sequence of the protein. The sequence containing labile amino acids such as Asn-Gly and Met would be indicative of potential instability issues. The rate of chemical reactions that alter the primary sequence of the protein is higher in solution conditions and can limit the shelf-life of protein therapeutics. As the mobility of reactants is minimized in the solid state, freeze-drying is often attempted to improve the stability [16]. In such instances, physical instability is a major issue to be dealt with. Freezedrying, also termed as lyophilization, is a dessication process in which the solvent (usually water) is first frozen and then is removed by sublimation in a vacuum [17]. In other words, the protein in solution is frozen, producing discrete ice and solute crystals. The solid ice is sublimed. Controlled heating desorbs any of the tightly bound water. 

Preparation of samples for use in RRAs varies. If the substance of interest is in biological fluids (e.g., urine, spinal fluid, plasma) it may be possible to add fluid directly to the RRA. In many instances, however, components (salts, proteins) interfere with assay integrity. In this case, it is necessary to devise a preparation scheme suitable for use with the RRA and which does not alter the quantities of the substance of interest, or to incorporate an appropriate internal standard in the method (e.g., radioactive tracer amounts of the compound of interest preferably labeled with an isotope different from that incorporated in the ligand). In the case of natural product analysis, standard aqueous or organic extraction procedures are followed and the sample is freeze-dried or dried to provide the final preparation. Aqueous fractions can be redissolved in assay buffers whereas dimethylsulfoxide (DMSO) is a useful solvent for organic extracts since many RRAs tolerate < 1 % DMSO in the final assay. 

Integral or intrinsic proteins of the B. cannot be removed except by dissolution of the B. with organic solvents or detergents these proteins are responsible for the bumps observed in freeze-firactured B. preparations under the electron microscope. Peripheral or extrinsic proteins can be stripped from the B. by altered ionic conditions or extremes of pH. 

---