Co-purification

Crude protein sources containing one glycoprotein usually contain multiple glycoproteins. In most such instances, lectin-based affinity systems will result in the co-purification of several such glycoproteins. 

The advantage of such co-purification protocols is that the fully processed protein serving as the bait can allow interactions in a native environment and cellular location to allow isolation of multicomponent complexes. One limitation with this approach is the necessity for an antibody with specific immunoreactivity and immunoprecipitative capability for the bait protein. This drawback can be addressed by expression of the protein with an epitope tag. Excellent antibodies to a variety of epitope tags are available and can be utilized for immunoaffinity purification. Tags such as 6-histidine and GST allow purification using affinity characteristics to nickel and GSH beads, respectively. 

T. M. Guenthner, D. Cai, R. Wallin, Co-Purification of Microsomal Epoxide Hydrolase with the Warfarin-Sensitive Vitamin Kx Oxide Reductase of the Vitamin K Cycle , Biochem. Pharmacol. 1998, 55, 169 - 175. 

Majerus and co-workers have published work on the co-purification of inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) with the CSN from bovine brain [24,... 

The TC biodiesel fuel processor instead consists of a two-reactor system, in which one reactor, the cracker, is used for production, while the other one is being regenerated by gasification of the deposited sohd C with steam yielding H, COj, CO and CH (Ledjefif-Hey et al., 2000). The product gas streams of the TC and the gasification unit are combined, cooled down to the shift inlet temperature and then fed to the shift reactor and the CO-purification step. First, the cracking of biodiesel takes place for the production of H ... 

H2A.Bbd was discovered as an H2A-like protein encoded by human ESTs [77]. H2A.Bbd is only 42% identical to conventional H2A and is smaller due to a shortened C-terminus that lacks the ubiquitination site that is present in most other H2As (Fig. 6). H2A.Bbd appears to be associated with nucleosomes as indicated by the co-purification of an epitope-tagged H2A.Bbd with nucleosomal fragments in a sucrose gradient run at high ionic strength [77]. 

Azevedo, R.A. Smith, R.J. Lea, P.J. Aspartate kinase regulation in maize Evidence for co-purification of threonine-sensitive aspartate kinase and homoserine dehydrogenase. Phytochemistry, 31, 3731-3734 (1992)... 

Tamiya, N. Yusa, T. Yamaguchi, Y Tsukifuji, R. Kuroiwa, N. Moriyama, Y. Fujimura, S. Co-purification of thymidylate kinase and cytosolic thymidine kinase from human term placenta by affinity chromatography. Biochim. Biophys. Acta, 995, 28-35 (1989)... 

Cell-protecting additives, such as Pluronic F-68, are commonly included as a media component to reduce cell damage in gas-sparged, agitated cultures. Despite the value of Pluronic F-68 in protecting cells, there is a potential problem in that Pluronic may have some cytosolic effects it may reduce the yield of the producer cell line in culture and there is a possibility of complexation or co-purification with a cell product. The replacement of Pluronic with fatty acids, as well as being positive for the half-lives and production of cultured cell lines, provides an alternative method to protect producer cells in agitated cultures (Butler et al., 1999). 

Cohen, S., Carpenter, G., and King, L., Jr. (1980). Epidermal growth factor-receptor-protein kinase interactions. Co-purification of receptor and epidermal growth factor-enhanced phosphorylation activity. J. Biol. Chem. 255, 4834-4842. 

Song, K.S., Li, S., Okamoto, T., Quilliam, L.A., Saigiacomo, M. and Lisanti, M.P. (1996) Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains. J. Biol. Chem. 271, 9690-9697. 

Cellobiase. Figure 2 outlines the general purification steps used to isolate a pure cellobiase (named for its function in the cellulase complex) and three forms of the hydrocellulase. In purifying cellobiase it was expedient to replace the adsorption or affinity column with a batch separation on DEAE-Sephadex A-50 and to complete the purification with cation exchange chromatography on SP-Sephadex (49, 50). Table IV is a summary of the purification of the cellobiase and co-purification of... 

Song KS, Li S, Okamoto T, QuilUam LA, Sargiacomo M, Lisanti MP. Co-purification and direct interaction of Ras with cave-olin, an integral membrane protein of caveolae microdomains. 

Figure 4 Elution of the NEM modified Cys from the PTH-analyzer. Cycles 4 to 6 of peak 2 (see Results and Discussion) are shown. Peak 2 shows the co-purification of 2 peptides the expected sequences in cycles 4 to 6 were...

Sumitomo Chemical Co. Purification of Fatty Acid Derivatives 1967 GB 1,074,693... 

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... 

Kuno, T., Andresen, J. W., Kamisake, Y., Waldman, S. A., Chang, L. Y., Saheki, S., Leitman, D. C., Nakane, M., and Murad, F. (1986). Co-purification of an atrial natriuretic factor receptor and particulate guanylate cyclase from rat lung. J. Biol. Chem. 261,5817-5823. 

For the CO purification in fuel-cell heating appliances were different processes proposed. As physical purification processes the pressure swing adsorption and membrane filter were proposed. Both process technologies are state of the art in the... 

High enzyme cost Secretory enzymes High-expression host High-cell-density fermentation Simple enzyme purification techniques Heat precipitation Ammonia sulfate precipitation Simple adsorption and immobilization Resin-free chromatographic separation Multi-enzyme co-immobilization and co-purification... 

Co-purification Assays to Measure Protein-Protein Interactions... 

For example, MBP and GST fusion proteins may be tested by mixing the respective crude lysates, purifying the MBP fusion protein using amylose resin, and assessing co-purification by immunoblot analysis using anti-GST primary antibodies. 

Downstream applications using HH-tagged proteins require additional precautions at this point. Thus, if protease inhibitors are included in the purification buffer used to resuspend cells, care must be taken to use inhibitors that are compatible with the His-Select affinity matrix. In particular EDTA used to inhibit metalloproteases can strip the affinity gel of cobalt. Including low concentrations of imidazole in the purification and wash buffers can help to prevent co-purification of undesirable proteins. Test with low concentrations (5-20 mM) first, and then increase if necessary. 

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