Agarose columns

Lipoproteins (from human plasma). Individual human plasma lipid peaks were removed from plasma by ultracentrifugation, then separated and purified by agarose-column chromatography. Fractions were characterised immunologically, chemically, electrophoretically and by electron microscopy. [Rudel et al. Biochem J 13 89 1974.]... 

Phosphoproteins (various). Purified by adsorbing onto an iminodiacetic acid substituted agarose column to which was bound ferric ions. This chelate complex acted as a selective immobilised metal affinity adsorbent for phosphoproteins. [Muszyfiska et al. Biochemistry 25 6850 1986.]... 

Serum samples from some animals fed the four dietary regimens were pooled and subjected to fractionation of the lipoproteins by agarose column chromatography. The total amounts of lipoproteins found in the sera from the four groups (ug/ml) were casein, 904 soy protein, 807 casein/arginine, 1,130 and, soy/lysine, 672. 

V-1 from acid and alkaline hydrolyzates, SCX-HPLC of amino acids, a mixture of purified crosslinks and hydroxylysine b purified cross-link V-2 c amino acids from an acid hydrolyzate (6 M HCl) of reduced bovine dentin retained on a phenylboronate agarose column after purification as high molecular weight fractions by repeated size exclusion chromatography d as c, alkaline hydrolyzate (2 M KOH). Injections (c, d) resulted from 18 and 52 mg collagen originally hydrolyzed, respectively. 1 = 111 (HP) 2 = V-2 3 = IV 4 = V-1-1 (DHLNL) 5 = HLNL (bovine tendon) 6 = VI (histidinoalanine ) 7 = hydroxylysine 8 = VI (lysinoalanine). 

Figure 4. Elution profile of the 2 - and 5 -0-glucosyltransferases after chromatography on Brown 10X agarose column using pH-salt gradient. Insert autoradiographed enzyme reaction products.

Polypeptide Chain Molecular Weight Determination by Gel Permeation Studies on Agarose Columns in 6M Guanidinium Chloride... 

B 9. Describe how you would use affinity elution to remove the enzyme alcohol dehydrogenase bound to a Cibracron blue-agarose column. 

Figure 3 Silver stained SDS-PAGE gel of iPLA2-VI purification. Lane 1, molecular mass markers lane 2,20 pi of pool from octyl-Sepharose column lane 3,20 pi of fraction 28 from the Calmodulin-agarose column lane 4,20 pi of fraction 29 from the Calmodulin-agarose column and lane 5,20 pi of fraction 30 from the Calmodulin-agarose column. (From Ref. 41.)...

The overall recovery of the enzyme was >60 % with a 235-fold purification. The final preparation, however, was not homogenous. Barley has two isoforms of ALS which can be separated on a phenyl agarose column immediately after the ammonium sulfate precipitation. One of the forms does not bind to the column and was too unstable to attempt purification. The details of purification in Table III pertain to the fraction with affinity for phenyl agarose. 

Figure 7-11. Purification ot staphylococcal nuclease by affinity adsorption chromatography on a nuclease-specific agarose column (0.8 x 5 cm). The column was equilibrated with 50mM borate buffer, pH 8.0, containing lOmAf CaClj. Approximately 50 mg of partially purified material containing about 8 mg nuclease was applied in 3.2 ml of the same buffer. After 50 ml of buffer had passed through the column, O.IM acetic acid was added to elute the enzyme. 8.2 mg nuclease and all the original activity was recovered. The flow rate was about 70 ml/hour. [From P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Natl. Acad. Sci. US, 61 636 (1968).]...

A novel method for the purification of trypsin was described by Wilchek and Goreki [66] and is based on the isolation of trypsin as a soluble complex with dinitrophenylated soybean trypsin inhibitor on an antidinitrophenyl antibody-agarose column. The procedure involved the dinitrophenylation of soybean trypsin inhibitor with dinitrobenzenesul-phonate, the formation of a complex between trypsin and the DNP-trypsin inhibitor within the sample mixture and adsorption of this complex to the anti-DNP-agarose column. The complex is then eluted, dissociated and pure trypsin recovered, for example, by gel filtration. The authors suggested a general applicability for this technique to all systems comprising two or more components. 

Using a method based on the oxamate-agarose column of O Cara and Barry, it has been found possible to separate the LDH-X isoenzyme from the other LDH isoenzyme in mouse testes [76]. Unlike the other isoenzymes, LDH-X had no affinity for the column. 

Eldefrawi and Eldefrawi [98] reported the purification of the acetylcholine of Torpedo electroplax on an affinity column consisting of cobra (Naja naja siamensis) toxin coupled to Sepharose 4B. Desorption with 10 mM benzoquinonium produced a protein that bound [ I]a-bungarotoxin but not [ H]acetyl-choline. However, desorption with 1 mM carbamylcholine gave a receptor protein that bound pH]acetylcholine decamethonium, [ H]nicotine [ C]dimethyl-(-l-)-tubocuranrine, and [ I]a bungarotoxin. Schmidt and Raftery [99] also purified acetylcholine receptor, from Narcine, on a N-(e-aminohexanoyl)-3-aminopropyltrimethyl-ammonium bromide-HBr-agarose column. 

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