Fractioning protein conjugates

FIGURE 4.49 Isolation of a complex protein conjugate on Toyopearl HW-50S. Column 22 mm X 83 cm. Sample Fraction from crude Tetrahymena H2A containing the ubiquitin-histone conjugate uH2A. Elution 10 nM HCI. Flow rate 0.1 ml/min. Detection UV at 230 nm. 

From a chemical point of view, the El/ubiquitin thiol ester should be competent to donate ubiquitin to a substrate amino group. In fact, aminoacyl-errzyme thiol esters are used in exactly this way in non-ribosomal polypeptide synthesis, a process that was discovered around the same time as ubiquitin-protein conjugation [5]. In spite of the attractive simplicity of this model, however, biochemical reconstitution studies showed that besides El two additional fractions were required to conjugate ubiquitin to a model substrate. They were called ubiquitin carrier protein (E2) and ubiquitin-protein ligase (E3), respectively, since the respective factors seemed to act sequentially [6]. Interestingly, the E2 factor apparently formed a thiol ester with ubiquitin. Based on these results, Hershko and co-workers proposed the ubiquitin conjugation cascade (Figure 5.1). 

Pedron et al generated ABA-protein (ovalbumin or BSA) conjugates through the C-1 carboxyl group or the C-4 carbonyl group of ABA (11 and 12) [20]. ELISA detection showed that these ABA-protein conjugates bound efficiently to the solubilized microsomal protein fraction of Arabidopsis thaliana, independent of the nature of the carrier protein or the ABA-carrier protein linker. Purification of these binding proteins has not yet been reported. 

Zhang, D. W, and Zhang, J. Z. H. (2003). Molecular fractionation with conjugate caps for full quantum mechanical calculation of protein-molecule interaction energy. The Joumai of Chemicai Physics 119,7, pp. 3599-3605. 

In order to analyze individual mono(ADPR) proteins we first set out to purify the acceptor(s) of the mitochondrial system which according to the subcellular distribution in liver represent a major fraction of this type of conjugate [10]. In the course of these studies evidence for a nonenzymic formation of mitochondrial ADPR protein conjugates accumulated [11]. 

Symmetrical flow FTP has Ireen successfully used to analyze numerous purified proteins and their dimers, protein conjugates, sodium dodecyl sulfate (SDS)-protein complexes, including precipitate and other biological samples, as summarized in Table 1. In these applications, sample pretreatment was not required even when the proteins of interest were present in complex matrices such as plasma, dairy products, and wheat flour (in contrast to SF3C and electrophoresis). Ultracentrifugation, which is commonly used to fractionate these samples, requires over 24 hr in comparison... 

Figure 15.9 The results of capture ELISA on native RNase A and formalin-treated RNase A. Right panel, native RNase A (curve 1) and unfractionated formalin-treated RNase A (curve 2). Left panel, individual fractions of formalin-treated RNase A monomer (curve 3), dimmer (curve 4), trimer (curve 5), tetramer (curve 6), and a mixture of oligomers with >5 cross-linked proteins (curve 7). The ELISA plate wells were coated with monoclonal antibody against bovine pancreatic RNase A (1 pg/mL) overnight at 4°C and then blocked with bovine serum albumin. The wells were incubated for lh at 37°C in the presence of various concentrations of antigen in lOOpL of PBS. After washing, each plate well received a 1 4000 dilution of horseradish peroxidase conjugated rabbit polyclonal anti-RNase A antibody followed by incubation at ambient temperature for lh. After washing, detection was achieved using a mixture of 2,2,-azino-di-(3-ethylbenzthiazoline-6-sulphonate) and hydrogen peroxide. Absorbance was monitored at 405 nm. See Rait etal.11 for details.

Pool the fractions containing protein. Adjust the enzyme concentration to lOmg/ml for the conjugation step (see next section). The periodate-activated enzyme may be stored frozen or freeze-dried for extended periods without loss of activity. Do not store the preparation in solution at room temperature or 4°C, since precipitation will occur over time due to self-polymerization. 

Pool the fractions containing protein. Adjust the antibody concentration to lOmg/ml for the conjugation step. The oxidized antibody should be used immediately. 

Elute the bound conjugate with 0.1M sodium acetate, 0.5M NaCl, pH 5.0. Pool the fractions containing protein, and dialyze the conjugate into lOmM sodium phosphate, 0.15 M NaCl, pH 7.0, or other suitable storage buffers. 

Alternatively, GFP can be visualized using rabbit polyclonal antibody raised against GFP purified directly from A. victoria. This anti-GFP antibody facilitates the detection of native GFP, recombinant GFP, and GFP-fusion proteins both by immunofluorescence and brightfield microscopy, as well as by western blot analysis and immunoprecipitation. Direct anti-GFP conjugates made from a complete serum or from an IgG fraction are available from Invitrogen (http //www.invitrogen.com/ site/us/en/home.html). Additional options for your research offered by Invitrogen include two mouse monoclonal antibodies and a chicken IgY fraction. 

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