Mono Q anion-exchange chromatography

Nunoi and co-workers (1988) fractionated neutrophil cytoplasm by Mono Q anion-exchange chromatography and obtained three fractions (NCF-1, -2 and -3) that were active in the assembly of the oxidase. Independently, Volpp and colleagues (Volpp, Nauseef Clark, 1988) prepared antiserum from cytosolic factors that eluted from a GTP-affinity column, and this antiserum (Bl) recognised cytoplasmic factors of relative molecular masses 47 kDa and 66 kDa. It was later shown by this group that these cytosolic factors translocated to the plasma membrane during activation. NCF-1 was shown to contain the 47-kDa protein and NCF-2 the 66-kDa protein. Analysis of the defect in the cytosol of autosomal recessive CGD patients revealed that most of these (88%) lacked the 47-kDa protein (p41 -phox), whereas the remainder lacked the 66-kDa protein (p66-phox). Both of these components have now been cloned and recombinant proteins expressed. Interestingly, in the cell-free system, recombinant p47-phox and p66-phox can restore oxidase activity of the cytosol from autosomal recessive CGD patients who lack these components. 

In our experience, PAPS from commercial sources was generally about 80% pure. However, significantly lower purities have been observed in some cases. A major impurity in any PAPS preparation is 3 -phosphoadenosine-5 -phosphate (PAP), which is formed by spontaneous hydrolysis of PAPS. As a by-product of the TPST-catalyzed sulfotransfer reaction, PAP acts as a product inhibitor of tyrosine sulfation (Danan, Yu, Hoffhines, Moore, Leary, 2008 Danan et al., 2010), which is one reason why sulfation rates of in vitro sulfation reactions decrease over time. Thus, for efficient in vitro sulfation of peptide substrates, in particular, if multiple sulfation sites are present, high-purity (>80%) PAPS preparations with low (<10%) PAP content are to be used. For these reasons, we found it necessary to routinely analyze aU PAPS preparations by ion-pair HPLC, and to repurify low-purity PAPS preparations by Mono Q anion-exchange chromatography. Furthermore, to minimize the formation of PAP, PAPS preparations should be kept at neutral pH and at very low temperatures (-80 °C). 

Purification of PAPS by Mono Q Anion-Exchange Chromatography (Optional)... 

Optionally, if PAPS purity is less than 80% or PAP content exceeds 10%, PAPS can be purified by Mono Q anion-exchange chromatography (Burkart, Izumi, Chapman, Lin, Wong, 2000). 

Buffer B 20 mM NaH2P04, pH7.5 0.5 M imidazole 1 M NaCl 1 mM MgOAc 5 mM /3-mercaptoethanol Mono Q anion exchange chromatography ... 

Mono Q Anion Exchange Chromatography. The desired fractions from the HiTrap column shonld be pooled and diluted in Mono Q Buffer A (Table IV) to a final volnme of 50 mL. This will dilute the salt concentration... 

In an attempt to learn more about its properties, the L-aminoamidase from M. neoaurum ATCC 25795, which was responsible for the enantioselective resolution of DL-a-methyl-valine amide, has been purified and characterized [1,49]. The purification procedure included ammonium sulfate fractionation, Superdex 200 gel filtration, and Mono Q anion exchange chromatography. By this procedure, the L-aminoamidase can be purified from the crude extract approximately 280-fold and with a 10% yield. The obtained enzyme preparation is homogeneous as judged by SDS-PAGE. 

Thirty-two years later, Ko jima etal. (2000a) purified Latia luciferase by the following steps ammonium sulfate fractionation, gel-filtration, affinity chromatography and Mono-Q anion-exchange FPLC. 

Mono-Q anion-exchange FPLC chromatography column. 

Anion Exchange. This purification is in the absence of calcium using a 5/5 Mono-Q anion-exchange FPLC chromatography column. 

Several modifications of the above protocol have been described for eco purification. These are very useful for the purification of eco variants as they consist of a gentler series of purification steps. Following the acidification step, the protein is dialyzed into distilled water and subjected to a 65% (w/v) ammonium sulfate precipitation. The insoluble fraction is resuspended in distilled water, dialyzed into 10 mM Tris pH 8.0, and concentrated. The resulting protein is purified to homogeneity by FPLC chromatography on a Mono-Q anion exchange column (Pharmacia) with a slow gradient of 0-15% NaCl. 

Clezardin, P., McGregorJ. L., Manach, M.,Boukerche, H., arid Dechavanne, M (1985) One-step procedure for the rapid isolatiort of mouse monoclonal antibodies and their antigen binding fragments by fast protein liquid chromatography on a Mono-Q anion exchange column./ Chromatogmphy S19, b - . 

Chromatography matrices. DEAE-cellulose (DE-52, Whatman) hydroxylapatite (Bio-gel HTP, Bio-Rad), Mono Q anion exchange columns (Pharmacia Fine Chemicals) and Ni-NTA nickel affinity chromatography support (Qiagen)... 

Purity Criteria. Purity of lignin peroxidase was analyzed with analytical anion exchange chromatography using detection at 405 nm. About 4 U of lignin peroxidase was applied to a Pharmacia Mono Q column (HR 5/5,0.25 x 5.0 cm) and eluted with linear sodium acetate, pH 6.0, gradient (0.01 to 0.55 M, 1 ml/min, 14 min). 

Recoverin was purified further by anion exchange chromatography using a Mono Q column (Figure 2, lane d). The purification of recoverin was accomplished using a linear gradient of NaQ (0-250 mM), and recoverin eluted at approximately 125 mM NaQ. 

Figure 7.7 Anion Exchange Chromatography of the proteins from the free run juice of grape of cv. Incrocio Manzoni 6.0.13. Column Tricorn Mono Q 5/50 GLM detection at wavelength 280 nm buffer A Tris-HCl 20 mM pH 8.5 buffer B buffer A containing NaCl 1 M gradient program from 0 to 14% of B in 70 min, from 14 to 100% B in 20min and from 100 to 0% B in 5min flow rate 0.6mL/min (Vincenzi et al., 2005)...

To determine whether HCS activity from pea leaves could be resolved into several forms by anion-exchange chromatography, extracts from pea leaves (crude leaf extract, chloroplast stroma, mitochondrial matrix and cytosol) were fractionated on a Mono Q HR 5/5 column [11]. We have observed that HCS activity from a crude extract could be resolved into two peaks. The minor peak, eluting at 50 mM NaCl represented approximately 10% of the total HCS activity and was detected in the chloroplast and mitochondrial fractions. The major peak (90% of the total activity), eluting at 140 mM NaCl was specific of the cytosolic fraction. These results strongly suggest the existence of two HCS isoforms, one localized in the cytosol and the other one in both chloroplasts and mitochondria. Thus, the occurrence of HCS isoforms in different cell compartments also suggest that the different biotin-dependent carboxylases present in plants are biotinylated in the cell compartment where these enzymes are active. 

Final purification of CRABP I by anion-exchange chromatography omit step 4 of Subheading 3.2. and concentrate the CRABP I-contammg fraction to 2 mg protein/mL with an Amicon concentrator/type YM5 filter while exchanging buffer to buffer B Apply five 1-mL aliquots sequentially to a Mono-Q column type HR5/5 and follow each application with a 5-min wash at 1 mL/mm with buffer B After the last wash, elute CRABP I at 1 mL/min with a linear gradient to 250 mM NaCl m buffer B over 25 mm (see Note 4)... 

Table 7. Purification of alcohol dehydrogenase from Lactobacillus kefir. (Phenyl-and octylsepharoses are materials for hydrophobic interaction chromatography Mono Q is an anionic exchanger.)...

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