Polyclonal antibody fractionation

Immuno localization using polyclonal antibodies raised against AE showed that AE was widely distributed in orange fruit but with more intensive immunological detection in the outer part of the peels e.g. albedo and flavedo and in the segments (juice vesicles). The results indicate that AE is located at the site where the major fraction of pectin is deposited. 

Polyclonal Antibodies against FORL r purified polygalacturonase were raised in white rabbits. For the first immunization 200 jxg of purified protein in 300 jxl of distilled water was mixed with 200 1 of PBS and 500 n of complete Freund s adjuvcmt and injected intramusculary into the leg. Two subsequent intramuscular injections, each containing 300 fig of protein in 1 ml of incomplete Freund s adjuvant were given at 1 month intervals. Finally, the rabbit was bled 1 week later. The antisera, separated from blood by incubation at 37 "C, were stored in 1 ml fractions at -20 C. 

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. 

In order to investigate whether tomatinases from F. oxysporum and F. solani share similar molecular characteristics, F. solani tomatinase was partially purified. Comparative SDS-PAGE analysis of the protein fractions with and without tomatinase activity showed the presence of a 32.5 kDa band in all positive fractions, while this band was absent in fractions without tomatinase activity The apparent molecular mass of tomatinase of F. solani differs from that of F. oxysporum (50 kDa), S. lycopersici (110 kDa) [33], and Botrytis cinerea (70 kDa)[36]. The F. solani tomatinase presents a very low activity compared with F. oxysporum enzyme [35, 89]. Western blot analysis showed that the two enzymes also differ in their immunological characteristics since the polyclonal antibody against tomatinase of F. oxysporum f. sp. lycopersici did not recognize the tomatinase from F. solani. These results suggest that the enzyme from F. solani is a novel tomatinase species. 

Oatpla3 consists of two variants (Oat-kl and Oat-k2) in the kidney (69,70). Oat-k2 lacks 172 amino acids at the amino terminal (70). The localization of Oat-kl has been suggested to be brush border membrane of the renal tubules since polyclonal antibody detected Oat-kl only in the brush border membrane-enriched fraction from the kidney (71). In contrast to other Oatps, Oat-kl mediates facilitated transport since the uptake by Oat-kl was insensitive to an ATP depleter (sodium azide) (69). Oat-kl accepts only folate derivatives such as MTX and folate, while the substrates of Oat-k2 include TCA and prostaglandin E2 in addition to these folate derivatives (69,70). 

E3. Endo, Y., Miyai, K., Hata, N., and Ichihara, K., Fractionation of polyclonal antibody by isoelectric focusing. Differences in cross-reactivity and affinity of rabbit donotype antihuman thyrotropin antibody. Anal. Biochem. 143, 249-255(1984). 

For polyclonal antibodies, negative reagent controls should be a dilution of immunoglobulin fractions or whole serum of normal/ non-immune serum of the same animal source. Again, the negative reagent control should be applied in the same concentration as the test antibody, and the same diluent should be used. 

Polyclonal antibody Possible contamination or undesired antibody in the host Ig fraction. 

Figure 6 illustrates the progressive overloading of an anti-HSA polyclonal antibody column after repeated injections of 2 /zg of HSA. At first injections, impurities elute from the column at the dead volume, while HSA is totally adsorbed. The gradual emergence of the nonretained HSA elution peak is due to two different effects, the saturation of the support and the slow adsorption kinetic process. The unretained fraction is calculated from peak area measurements, subtracting the area of the impurity response peak. 

The free amide peptide was synthesized on 4-methylbenzhydrylamine-substituted polystyrene resin, and the resin-bound peptide for antibody production was prepar on aminomethyl polystyrene. Cleavage/deprotection was done with hydrogen fluoride/anisole at 0 C. The free amide was not active in either bioassay. However, polyclonal antibody prepared against the resin-bound (22 24) sequence was immunologically reactive to EDNH reverse phase column fractions. We are now attempting to determine if the sequence represents an inactive fragment of authentic EDNH. 

Immnnoassays can also be combined with ply sicochemical methods such as HPLC, whereby the HPLC method separates the microcystins according to their hydrophobicity and the resulting fractions are analyzed by immunoassay. Immunoaffinity columns containing microcystin antibodies (Rivasseau 1999a) can also be used to confer specificity to the PPIA for the analysis of the cyanobac-terial toxins. Lin and Chu (1994) reported that polyclonal antibodies raised against microcy stin-LR could successfully be used to protect PP2 A from the action of microcystin in vitro. 

Platonova GA, Pankova GA, ITina lY, Vlasov GP, and Tennikova TB. Quantitative fast fractionation of a pool of polyclonal antibodies by immunoaffinity membrane chromatography. J. Chromatogr. A 1999 852 129-140. 

Polyclonal Antibodies After an antigen is injected into an animal by a regimen designed to induce an optimal immune response, serum can be collected from the animal and the immunoglobulin fraction isolated. This antisera is enriched with antibodies specific for the original antigen. Because a large number of lymphocytes are involved in the production of the antisera, antibodies produced by this classical method are called polyclonal. 

Polyclonal antibodies can be used as whole serrim or in purified form, the latter either as the total immunoglobulin fraction or aflinity purified. Due to the heterogeneity of the antibody-binding sites of polyclonals, different selectivity for different antigens will appear and this problem is simply avoided by the use of monoclonal antibodies. However, even though it is often... 

Figure 2 Recombinant expression of human mEH protects V79 Chinese hamster cells (V79 cells) from styrene-7,8-oxide-induced DNA single-strand breaks. (A) Western blot analysis of mEH transfected (lanes 1—6) and mock transfected (lane V) V79 cells. S9 fractions of the different clones were analyzed with a polyclonal antibody against purified human mEH raised in rabbits. Lane M prestained molecular weight markers (195, 112, 84, 63, 52, 35, and 32 kd). (73) mEH activity of the same clones using styrene-7,8-oxide as a substrate. Values are expressed as nanomoles of styrene glycol formed per minute and milligrams of protein. (C) Effect of styrene-7,8-oxide on DNA single-strand breaks using mEH transfected clone No. 3 [from (A) and (73)] and mock transfected V79 cells (7).

Reduction of Cu and Fe organic complexes is saturable, and is inhibited by cell-impermeable probes, including polyclonal antibodies raised to Chlorella nitrate reductase (Jones and Morel, 1987). The model proposed suggests that a fraction of a membrane-bound nitrate reductase (the so-called diaphorase component that catalyzes NADH reduction), spans the cell membrane (Fig. 4). Organic metal complexes intercept electrons (destined for nitrate reduction intracellularly) at the outer cell surface. 

KS Joshi, JE Butler. The immunochemistry of sandwich ELISAs. V. The capture antibody performance of polyclonal antibody-enriched fractions prepared by various methods. Mol Immunol 29 971, 1992. 

Fig, 1. Left Western blot stained with HCP polyclonal antibodies. Lane 1 contains mw markers (20, 30, 40, 50, 60, 80, 120, 220 kD), lane 2 is a sample of production fraction 2.1 diluted 250 times, lane 3 contains a sample of production fraction 3.1 diluted 10 times, lane 4 contains the HCP-mix diluted 10 times, lane 5 contains an undiluted sample of production fraction 5.1. Lane 6 contains normal rabbit serum as a negative control. Right, residual protein staining blot (right, lane 1 mw markers, lane 2 empty, lanes 3-9 correspond with lanes 1-6 of the western blot)... 

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