Affinity precipitation

Water-soluble polymers and polyelectrolytes (e.g., polyethylene glycol, polyethylene imine polyacrylic acid) have been used success-hilly in protein precipitations, and there has been some success in affinity precipitations wherein appropriate ligands attached to polymers can couple with the target proteins to enhance their aggregation. Protein precipitation can also be achieved using pH adjustment, since proteins generally exhibit their lowest solubility at their isoelectric point. Temperature variations at constant salt concentration allow for frac tional precipitation of proteins. 

Protein isolation with affinity precipitation has been discussed in detail by Mattiasson and co-workers (see, e.g. Galaev and Mattiassion, 1997) and they have provided an exhaustive tabulation. Polymers varied from alginate.s/chitosan to dextran to NIPAM. Examples of the used proteins are from trypsin, p-glucosidase, xylanase, alkaline protease, etc. It is remarkable that affinity precipitation can sometimes give results comparable to affinity chromatography. 

Lali A, Balan S, John R, D Souza F (1998), Carboxymethyl cellulose as a new heterobifunctional ligand carrier for affinity precipitation of proteins, Bioseparation 7 195-205. 

Extraction in aqueous two-phase combined with precipitation yielding a removal of cell debris in the extraction step, and elimination of most impurities in the affinity precipitation step... 

Senstad, C. Mattiasson, B. "Affinity-Precipitation Using Chitosan as Ligand Carrier" Biotech, and Bioeng. 1989, 33, 2, pp 216-220. 

Affinity Precipitation of Avidin by Using Ligand-Modified Surfactants... 

Schneider et (3) provided a concise list of the possible advantages of affinity precipitation in the large scale separation of biological molecules. 

Figure 1. Structural formulas of the ethoxylated alcohol and phospholipid surfactants used in the affinity precipitation of avidin. Octaethyleneglycol mono-n-dodecylether (C12E8) was used as a solubilizing surfactant and dimyristoylphosphatidylethanolamine (DMPE) was used as the insoluble surfactant to which biotin was covalently attached. (The structural formula of the derivatized phospholipid and a discussion of the reaction and purification scheme have been previously described by Powers et (7)).

Affinity Precipitation of Avidin from Partially Purified Egg Whites. Following the ion exchange fractionation of egg white solution, described in the experimental section above, fractions of eluted protein solution (obtained from the elution described in Figure 6) were mixed with... 

Figure 9. Continued, (c) Ultraviolet—visible spectrum of the supernatant after affinity precipitation of avidin from an avidin—myoglobin solution (both 10" M in 0.2 M ammonium carbonate buffer at pH 8.9) by a DMPE-B-C12E8 solution. The supernatant was sampled after precipitation for two hours and centrifugation at 5000 rpm for twenty minutes, (d) Ultraviolet—visible spectrum of avidin—DMPE-B precipitate described in text and caption 9(c) after resuspension in 10" M C12E8 solution in 0.2 M ammonium carbonate buffer at pH 8.9.

The use of a novel affinity precipitation scheme, applicable to multibinding site biomolecules, has been illustrated by the purification of avidin from model protein solutions and from partially purified egg whites. The... 

Figure 10. Continued. Reverse phase HPLC chromatograms of (c) supernatant of Fraction B following affinity precipitation and (d) reconstituted avidin from egg whites following affinity precipitation.

Figure ll. SDS-PAGE patterns from (a) standard Vector Labs avidin solution, (b) Fraction B from ion-exchanged partially purified egg whites before affinity precipitation (described in experimental section), (c) same as (b) but after affinity precipitation, and (d) purified avidin from Fraction B after affinity precipitation and reconstitution. 

A direct method for testing RNA-protein interaction in vitro is by affinity-precipitation or -chromatography. Two molecules interact if specific purification of one of them leads to co-purification of the other molecule. This approach is often referred to as the pulldown method . Different experimental designs can be used One possibility is to let the RNA-protein complex form in solution under native conditions, and then purify the complex by binding a high affinity tag, present in either the RNA or the protein to beads. The physical separation may be accomplished by low speed centrifugation (batch approach) or by column chromatography (column... 

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