Slab Gel Isoelectric Focusing Research Articles

Surface isoelectric focusing (sIEF) with carrier ampholyte pH gradient

Isoelectric focusing (IEF) is a powerful tool for amphoteric protein separations because of high sensitivity, bio-compatibility, and reduced complexity compared to chromatography or mechanical separation techniques. IEF miniaturization is attractive because it enables rapid analysis, easier adaptation to point of care applications, and smaller sample demands. However, existing small-scale IEF tools have not yet been able to analyze single protein spots from array libraries, which are ubiquitous in many pharmaceutical discovery and screening protocols. Thus, we introduce an in situ, novel, miniaturized protein analysis approach that we have termed surface isoelectric focusing (sIEF). Low volume printed sIEF gels can be run at length scales of ∼300μm, utilize ∼0.9ng of protein with voltages below 10V. Further, the sIEF device platform is so simple that it can be integrated with protein library arrays to reduce cost; devices demonstrate reusability above 50 uses. An acrylamide monomer solution containing broad-range carrier ampholytes was microprinted with a Nano eNablerTM between micropatterned gold electrodes spaced 300μm apart on a glass slide. The acrylamide gel was polymerized in situ followed by protein loading via printed diffusional exchange. A pH gradient formed via carrier ampholyte stacking when electrodes were energized; the gradient was verified using ratiometric pH-sensitive FITC/TRITC dyes. Green fluorescent protein (GFP) and R-phycoerythrin (R-PE) were utilized both as pI markers and to test sIEF performance as a function of electric field strength and ampholyte concentration. Factors hampering sIEF included cathodic drift and pH gradient compression, but were reduced by co-printing non-ionic Synperonic® F-108 surfactant to reduce protein-gel interactions. sIEF gels achieved protein separations in <10min yielding bands < 50μm wide with peak capacities of ∼8 and minimum pI differences from 0.12 to 0.14. This new sIEF technique demonstrated comparable focusing at ∼100 times smaller dimensions than any previous IEF. Further, sample volumes required were reduced four orders of magnitude from 20μL for slab gel IEF to 0.002μL for sIEF. In summary, sIEF advantages include smaller volumes, reduced power consumption, and microchip surface accessibility to focused bands along with equivalent separation resolutions to prior IEF tools. These attributes position this new technology for rapid, in situ protein library analysis in clinical and pharmaceutical settings.

Heterogeneity of protein labeling with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde

Fluorogenic reagents are used for protein labeling when high-sensitivity fluorescence detection is required. Similar to traditional labeling with activated fluorescent dyes, such as fluorescein isothiocyanate, a fluorogenic reaction is expected to change the physical–chemical properties of proteins. Knowledge of these changes may be essential for efficient separation and identification of labeled proteins. Here we studied the effect of labeling of myoglobin with a fluorogenic reagent on the acid–base properties of the protein. The fluorogenic reagent used was 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). In slab-gel isoelectric focusing, we found that the labeling reaction generated at least six species with p I values lower than that of non-labeled myoglobin. These species can be identified as products of progressive labeling of myoglobin with one to six FQ molecules. The same series of FQ-labeled species were observed when the reaction products were analyzed by capillary zone electrophoresis. The comparison of experimental and theoretical p I values allowed us to elucidate the labeling pattern—the number of FQ molecules corresponding to each labeled product detected by isoelectric focusing.

Differential phosphorylation of PKR associates with deregulation of eIF-2alpha phosphorylation and altered growth characteristics in 3T3-F442A fibroblasts.

Murine embryonic 3T3-F442A fibroblasts contain elevated levels of a factor (dRF) inhibitory to the phosphorylation of PKR, when cultured under differentiation restrictive (10% cat serum) as compared to permissive conditions (10% fetal bovine serum). Experiments were conducted with the objective of understanding the effect of altered PKR activity on the growth characteristics of 3T3-F442A fibroblasts. Analysis of the phosphoprotein pattern confirmed that the phosphorylation of PKR was reduced in cells cultured in cat serum during specific stages of growth. In a similar manner, evaluation of eIF-2alpha phosphorylation by vertical slab gel iso-electric focusing indicated that inactivation of PKR correlated with reduction of eIF-2alpha phosphorylation. The expression of PKR was confirmed by western blotting ruling out the possibility of diminished protein as the cause of loss of activity. In addition, the expression of dRF coincided with the inactivation of PKR as shown by immunoblotting and phosphorylation studies. The reduction in PKR activity and subsequent deregulation of eIF-2alpha phosphorylation was related to appearance of tumor-like cellular morphology and increased cell density as shown by cell counts and [3H]-thymidine uptake. Taken together, these results support a hypothesis that PKR functions to regulate the growth of 3T3-F442A cells. Furthermore, our findings raise the possibility that deregulation of PKR by endogenous inhibitory molecules, such as dRF, may alter normal growth and differentiation. Such a deregulation of PKR may also contribute to the proliferation of tumor cells.

High-performance capillary electrophoresis for in-process control in the production of antithrombin III and human clotting factor IX

Antithrombin III (ATIII) and factor IX (FIX), two proteins from the clotting cascade, were investigated in parallel experiments, using capillary gel electrophoresis and capillary isoelectric focusing. The results from these experiments were compared with the results obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and slab gel isoelectric focusing. In the case of ATIII, capillary gel sieving showed comparable results to SDS-PAGE with the added advantage of the shorter time required for analysis. By optimizing capillary isoelectric focusing (cIEF), a separation of the ATIII isoforms was achieved. In the case of FIX, capillary gel electrophoresis (SDS-CE) of a FIX preparation gave similar results to those obtained by size-exclusion high-performance liquid chromatography and SDS-PAGE, but turned out to be less sensitive in detecting protein impurities at low concentrations. The microheterogeneity of this protein was shown by using cIEF.

Current trends in capillary isoelectric focusing of proteins

Isoelectric focusing (IEF) in thin capillaries is reviewed here. After an introduction on the genesis and chemistry of the carrier ampholyte buffers, different approaches to IEF are discussed and evaluated. The classical approach consists on IEF under conditions of suppressed electroosmotic (EOF) flow, usually obtained by covalently bonding hydrophilic polymers to the inner capillary wall. The other approach consists of IEF in dynamically (and partially) coated capillaries, so as to allow a reduced EOF flow to coexist with the IEF process, so that focusing and transport of the train of stacked bands occurs simultaneously. The various experimental parameters: focusing, elution and detection steps, p I measurements, as well as typical drawbacks, such as isoelectric precipitation are evaluated. The review ends with some examples of analytical separations, at the moment mostlyl limited to focusing of native hemoglobins (normal and point mutants). These separations are compared with those obtained by slab-gel IEF and in immobilized pH gradients.

Electrophoretic separation of recombinant tissue-type plasminogen activator glycoforms: validation issues for capillary isoelectric focusing methods.

Attempts were made to validate a capillary isoelectric focusing (cIEF) method for a recombinant glycoprotein as an alternative technique to slab gel isoelectric focusing methods routinely used to monitor such charge heterogeneity. The cIEF method principally separates the charged glycoforms of recombinant tissue-type plasminogen activator (rt-PA) on the basis of their sialic acid content. Nine to ten distinct peaks were consistently resolved, with the profile dependent on the class of ampholyte used. The pI of rt-PA measured with synthetic pI standards was in the range pH 6.5-7.5 with the migration of the standards affected by the presence of the protein. The method showed an acceptable recovery of > 100% and had good sensitivity where 25 ng of protein could be resolved into constituent peaks. Recovery of both major peaks and total protein measured by peak areas was linear over a wide range from 50-1000 micrograms/mL. A detailed study showed that when a capillary had been used for some time, capillary age affected peak migration times and, to a lesser extent, resolution. Peak migration times were stable over a temperature range of 15-30 degrees C, and decreased predictably with increasing voltages (400-600 V/cm) and decreasing N,N,N',N'-tetramethylethylene diamine (TEMED) concentrations (0.4-1.5% v/v). Overall the data indicated that this methodology has the potential to be used in the commercial release of protein pharmaceuticals if variability resulting from capillary age and lot were resolved. Even in its present format the method equals the performance of slab gel IEF whilst offering significant improvements in ease of operation and in time and reagent use.

Rapid one-step capillary isoelectric focusing method to monitor charged glycoforms of recombinant human tissue-type plasminogen activator

A rapid (<10 min) one-step capillary isoelectric focusing (cIEF) method was developed to monitor charged glycoforms of recombinant human tissue-type plasminogen activator (rt-PA). Focusing takes place between the detector and the anode and the electro-osmotic flow (EOF) sweeps the separated glycoforms past the detector, towards the cathode. The separation uses a neutral coated capillary and hydroxypropylmethylcellulose (HPMC) to reduce the EOF to a constant and reproducible value. The method uses an ampholyte mix with a 50:50 ratio of pH 5–8 and pH 3–10 ampholytes in 4 M urea and 0.1% HPMC to produce maximal resolution whilst maintaining protein solubility during focusing. The electropherograms were compared to isoelectric focusing (IEF) slab gels of samples of intact rt-PA. In both cases approximately ten charged species could be detected. Data analysis indicated that the intra-assay precision was < 5% for peak migration times and < 10% for normalized peak areas. The number of charged species detected by each of the two methods was consistent for samples of intact rt-PA, rt-PA types I and II and for neuraminidase-digested rt-PA. Overall the data indicate that the automated cIEF method can be an adjunct to slab-gel IEF in the characterization and routine analysis of recombinant glycoproteins.

High-Resolution Slab Gel Isoelectric Focusing: Methods for Quantitative Electrophoretic Transfer and Immunodetection of Proteins as Applied to the Study of the Multiple Isoelectric Forms of Ornithine Decarboxylase

A high-resolution isoelectric focusing vertical slab gel method which can resolve proteins which differ by a single charge was developed and this method was applied to the study of the multiple isoelectric forms of ornithine decarboxylase. Separation of proteins at this high level of resolution was achieved by increasing the ampholyte concentration in the gels to 6%. Various lots of ampholytes, from the same or different commercial sources, differed significantly in their protein binding capacity. Ampholytes bound to proteins interfered both with the electrophoretic transfer of proteins from the gel to immunoblotting membranes and with the ability of antibodies to interact with proteins on the immunoblotting membranes. Increasing the amount of protein loaded into a gel lane also decreased the efficiency of the electrophoretic transfer and immunodetection. To overcome these problems, both gel washing and gel electrophoretic transfer protocols for disrupting the ampholyte-protein binding and enabling a quantitative electrophoretic transfer of proteins were developed. Two gel washing procedures, with either thiocyanate or borate buffers, and a two-step electrophoretic transfer method are described. The choice of which method to use to optimally disrupt the ampholyte-protein binding was found to vary with each lot of ampholytes employed.

Rat fibroblasts transfected with the human 70-kDa heat shock gene exhibit altered translation and eukaryotic initiation factor 2 alpha phosphorylation following heat shock.

Heat shock inhibits translation in a wide variety of cells. After heating, eukaryotic initiation factor 2-alpha (eIF-2 alpha) becomes phosphorylated which prevents the binding of Met-tRNA to the 40s ribosomal subunit inhibiting initiation of translation. Thermotolerant cells demonstrate resistance to inhibition of translation by additional heating suggesting that heat shock proteins may help to maintain translational integrity following thermal stress. Here we have examined the effects of increased intracellular levels of hsp70 protein on translation and eIF-2 alpha phosphorylation using rat fibroblasts stably transfected with a cloned human hsp70 gene. We observed a decrease in the rate of translational inhibition following heat shock in both hsp70-transfected and thermotolerant cells. Upon recovery at 37 degrees C, both hsp70-transfected and thermotolerant cells exhibit a faster rate of translational recovery. Utilizing slab gel isoelectric focusing coupled with immunoblotting we demonstrate that 45 degrees C heat shock leads to a rapid 4-5-fold increase in eIF-2 alpha phosphorylation, with little difference seen between control cells and hsp70-transfected cells. However, dephosphorylation of eIF-2 alpha occurs faster in the hsp70-transfected cells. These results suggest that hsp70 may play a role in facilitating the dephosphorylation of eIF-2 alpha as well as reversing the inhibition of translation following heat shock.

Analysis of a recombinant granulocyte macrophage colony stimulating factor dosage form by capillary electrophoresis, capillary isoelectric focusing and high-performance liquid chromatography

The analysis of a recombinant granulocyte macrophage colony stimulating factor (GM-CSF) dosage form by free solution capillary electrophoresis (FSCE), capillary isoelectric focusing (cIEF), and high-performance liquid chromatography (HPLC) is described. The quantitative use of capillary electrophoresis, whether FSCE or cIEF, will always be prone to special problems, such as sensitivity to differences in salt concentrations between the standard and sample, and can not match the ruggedness of HPLC. The usable quantitative linear range for both HPLC and FSCE surpass that achieved for cIEF methods by a factor of 10 or greater. The FSCE system, utilizing an octyl bonded/Brij-35 coated capillary, did not work for all proteins examined. This is probably due to an interaction of the protein with the bonded phase or the absorbed Brij-35. In contrast, the cIEF method worked well for all proteins tested thus far, yielding high efficiency and resolution comparable to slab gel isoelectric focusing. This paper addresses the potential for using free solution capillary electrophoresis and capillary isoelectric focusing as a quantitative analytical tool. Also, the effect of salt in the dosage form on quantitation, reproducibility, and efficiency of capillary electrophoresis methods is also discussed.

Polymer- and surfactant-coated capillaries for isoelectric focusing

This paper reports a method for deactivation of fused-silica capillaries to be used in capillary isoelectric focusing (cIEF). Deactivation was achieved by adsorbing either a surfactant or hydrophilic polymer to alkylsilane-derivatized capillaries. The surfactant PF-108 and methyl cellulose reduced electro-osmotic flow (EOF) 20 to 30 fold in comparison to underivatized capillaries. Although EOF was reduced sufficiently to allow focusing to permit separations to be completed before proteins were swept through the capillary, there was adequate flow to obviate the need for a separate mobilization step. This reduces the complexity of cIEF and increases reproducibility. Based on resolution of hemoglobin variants, proteins that varied 0.03 pH units in isoelectric point were resolvable. This is equivalent to the highest resolution achieved in conventional slab and tube gel isoelectric focusing.

Quantitation of proteins separated in N,N′-1,2-dihydroxyethylenebisacrylamide-crosslinkedpolyacrylamide gels

A simple and rapid method for the quantitation of proteins separated either by sodium dodecyl sulfate-electrophoresis or by isoelectric focusing in slab gels is presented. The method is based on the solubility of polyacrylamide gels crosslinked with N,N′-1,2-dihy-droxyethylenebisacrylamide (DHEBA) in periodic acid. After electrophoretic separation proteins are stained with Coomassie brilliant blue G-250. DHEBA gels show considerable swelling during the staining and destaining process but can be shrunk to their normal size in a 10% (w/v) solution of ammonium sulfate. Stained bands are cut from the gel and solubilized in periodic acid. During dissolution the dye decolorizes. Protein concentration in the solution is determined by a modified Coomassie dye-binding assay. Quantitation is linear in the range of 100 ng to 5 μg and not disturbed by dissolved gel. Separations in N,N′-1,2-dihydroxyethylenebisacrylamide-crosslinked gels show qualities similar to those in normal crosslinked gels.

Evaluation of protein phosphorylation state by a combination of vertical slab gel isoelectric focusing and immunoblotting

Conditions have been established for one-dimensional isoelectric focusing using vertical slab gel electrophoresis, followed by immunoblotting, for the measurement of the phosphorylation state of proteins. The method provides a less time-consuming alternative to two-dimensional gel electrophoresis combined with radiolabeling or immunoblotting. The main advantage of the method is that many samples can be analyzed simultaneously. The technique is applied here to the study of a mammalian initiation factor for protein synthesis, eukaryotic initiation factor 2 (eIF-2). The method allows good separation and quantitation of the different phosphorylated forms of the α subunit of eIF-2, when used to analyze either purified eIF-2 or eIF-2 contained in complex mixtures. The method is shown to be well adapted to the measurement of rapid phosphorylation/dephosphorylation kinetics in cell extracts, as well as the measurement of the phosphorylation state of eIF-2 in cultured cells. In addition, the method is shown to confirm the existence of a second phosphorylation site on eIF-2. Although eIF-2 has been used for this demonstration of the efficacy of the method, the technique is applicable to a study of the regulation of covalent modification of any polypeptide for which antibodies are available.

Detection of 10 variants of biliverdin reductase in rat liver by two-dimensional gel electrophoresis

We have identified and characterized multiple forms of biliverdin reductase (BVR) in control rat liver cytosol. Two-dimensional electrophoresis of the purified BVR resolved a minimum of 10 discrete protein zones. All 10 proteins were BVR as judged by immunological cross-reactivity toward rabbit anti-rat BVR. Based on the isoelectric focusing pattern of separation, the BVR variants could be organized into five net-charge groups designated as BVR-IEF1 to BVR-IEF5 and three molecular mass groups designated as BVR-MW1-BVR-MW3, respectively. The pI values of the net-charge groups were: BVR-IEF1, 6.23; IEF2, 5.91; IEF3, 5.76; IEF4, 5.61; IEF5, 5.48. The Mr values of the molecular mass groups were: BVR-MW1, 30,400; MW2, 30,700; MW3, 31,400. Single dimension slab gel isoelectric focusing offered greater resolution of the net charge variants, and BVR-IEF3 was further resolved into two variants, IEF3a and IEF3b, with pIs of 5.77 and 5.75, respectively. The six net-charge variants also resolved on a preparative chromatofocusing column and were designated as BVR-CF1-BVR-CF6. The pH values of the peak fractions were: BVR-CF1, 6.91; CF2, 6.33; CF3, 6.03; CF4, 5.82; CF5, 5.45; CF6, 5.27. Correspondence between the isoelectric focusing net-charge variants and the chromatofocusing net-charge variants was established. The Mr and net-charge variants did not represent partially degraded forms of biliverdin reductase produced during purification since the pattern of resolution of variants on slab gel isoelectric focusing or two-dimensional electrophoresis did not change by purifying the proteins in the presence of protease inhibitors and 5 mM EDTA. BVR-CF2 and BVR-CF4 were purified and examined for pH-dependent cofactor requirements for activity. Both net-charge variants and two pH optima that were cofactor-dependent; maximum activity with NADPH, however, was at pH 8.5 and with NADH at pH 6.7. With both variants, however, a higher catalytic rate was observed with NADH than with NADPH at their respective pH optima. Furthermore, BVR-CF2 exhibited a higher catalytic rate than did BVR-CF4 with either cofactor throughout the pH range of 5-9.

Apolipoprotein A-IV polymorphism and its effect on plasma lipid and apolipoprotein concentrations.

Recently, we determined the apolipoprotein E (apoE) phenotype distribution in 2,000 randomly selected 35-year-old male individuals by slab gel isoelectric focusing of delipidated plasma samples, followed by immunoblotting using anti-apoE antiserum. These blots have been successfully re-used for immunovisualization of apoA-IV isoelectric focusing patterns. In a population sample of 1,393 individuals, four distinct apoA-IV isoforms were detected, encoded by the alleles A-IV*0, A-IV*1, A-IV*2, and A-IV*3 with gene frequencies of 0.002, 0.901, 0.079, and 0.018, respectively. The mean of plasma cholesterol, triglyceride, apoB and E levels did not differ significantly among the different apoA-IV phenotype groups. For these lipoprotein parameters, less than 0.1% of the total phenotypic variance could be accounted for by the APOA-IV gene locus. Our results did not show any effect of apoA-IV polymorphism on plasma apoA-I levels nor could we find any correlation between plasma levels of apoA-I and apoA-IV within the different apoA-IV phenotype groups. The plasma level of apoA-IV in subjects bearing the A-IV*3 allele is significantly lower than in subjects without the A-IV*3 allele (5 mg/dl versus 14 mg/dl). We therefore conclude that, in contrast to the apoE polymorphism, the polymorphism at the APOA-IV locus does not influence any of the levels of the lipoprotein parameters considered except apoA-IV.

Differential binding of thyroxine and triiodothyronine to acidic isoforms of thyroid hormone binding globulin in human serum.

The differential availability of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) to liver from the circulating thyroid hormone binding globulin (TBG)-bound pool suggests that the two thyroid hormones may bind to different TBG isoforms in human serum. In the present study, the binding of [125I]T4 and [125I]T3 to human serum proteins was investigated by using slab gel isoelectric focusing and chromatofocusing. In normal human male serum, [125I]T4 was localized to four isoforms of TBG called TBG-I, -II, -III, and -IV, with isoelectric points (pI's) of 4.30, 4.35, 4.45, and 4.55, respectively. [125I]T3 was localized to only two isoforms of TBG, TBG-III and -IV, with pI's that were identical with those for [125I]T4. In normal female serum, [125I]T4 was localized to the same four isoforms of TBG as those of normal male serum, while [125I]T3 was localized to TBG-II, -III, -IV, and -V (pI = 4.65). In pregnant female serum, [125I]T4 was localized to five isoforms, whereas [125I]T3 was localized to four. IEF was also performed with male serum loaded with various concentrations of unlabeled T3. The Ki values of T3 binding to TBG-I, -II, -III, and -IV were 5.0, 2.4, 0.86, and 0.46 nM, respectively. The TBG isoforms in normal male serum were also separated by sequential concanavalin A-Sepharose affinity chromatography and chromatofocusing (pH range of 3.5-5.0). T4 preferentially bound to the most acidic isoforms of TBG in the pI range of 3.8-4.0, whereas the less acidic fractions (pH 4.0-4.2) bound both T4 and T3. In conclusion, this study shows that T4 and T3 do not bind to a single competitive binding site on TBG. Instead, T4 is preferentially bound by the most acidic TBG isoforms owing to a 10-fold lower affinity of T3 for these proteins.

Purification and properties of an acyl CoA transferase from Ascaris suum muscle mitochondria.

An acyl CoA transferase has been purified to electrophoretic homogeneity from the soluble compartment of Ascaris suum muscle mitochondria. From SDS-PAGE, isoelectric focusing and molecular exclusion chromatography, homogeneity was confirmed and the enzyme appears to be composed of two similar or identical subunits of apparent mol. wts of 50,000 resulting in an apparent mol. wt of 100,000 for the holoenzyme. The apparent isoelectric point was 5.6 +/- 0.1 by both chromatofocusing columns and slab gel isoelectric focusing. The transferase was relatively specific for the short, straight-chain acyl CoA donors as well as the CoA acceptors, being active on acetyl CoA, propionyl CoA, butyryl CoA, valeryl CoA and hexanoyl CoA as donors to acetate and propionate. Neither succinyl CoA nor succinate were appreciably active as CoA donor or acceptor, respectively. This enzyme cannot serve physiologically to activate succinate for decarboxylation to propionate, but may serve to ensure a supply of propionyl CoA which appears to be required in catalytic amounts for the decarboxylation of succinate.

Isoelectric focusing of the chick eye ciliary neuronotrophic factor.

A procedure is presented in which a crude extract from selected chick embryo intraocular tissues is submitted to analytical polyacrylamide slab gel isoelectric focusing. The extract contains a protein, ciliary neuronotrophic factor (CNTF) which can be eluted in active form from focused gels in a region occupied by only two protein bands. A "slot" technique is presented in which we demonstrate that the eluted CNTF activity focuses in the very restricted region between the two visible bands and is not associated with either band. Silver stain-densitometry is used to correlate staining intensity with protein concentration and from such an analysis it is concluded that the CNTF protein represents an extremely low proportion of total extract protein and that the minimum CNTF specific activity eluted from gel slices is 10(6) trophic units per mg protein. This one-step procedure will be used in the future to prepare highly purified CNTF for antibody generation.

Biochemical analysis of the p30's of N-, B-, and B leads to NB-tropic murine leukemia viruses of BALB/c origin.

Previous analysis of the virion proteins of an N- and a B-tropic type C virus of BALB/c mice, of 16 N-tropic recombinants (XLPN viruses) between these viruses, and of eight NB-tropic viruses derived from the B-tropic virus suggested that among these closely related viruses N-, B-, or NB-tropism was associated with the electrophoretic mobility of p30 on sodium dodecyl sulfate-polyacrylamide gels, and thus that p30 might determine this phenotype. To obtain further evidence for the association of structural markers of p30 with N-, B-, or NB-tropism, we have analyzed the p30's of these same viruses by using two-dimensional tryptic peptide mapping and slab gel isoelectric focusing. The results of these analyses suggest that (i) a single peptide unique to the N-tropic virus p30- is present in the p30 of all N-tropic recombinants; (ii) a single peptide unique to the B virus p30 is not present in p30's of the N-tropic recombinants, and this peptide is also absent in p30's of NB-tropic viruses derived from the B-tropic virus; and (iii) p30's of NB-tropic viruses possess a new tryptic peptide not found in the p30 of their B-tropic virus progenitors, and this new peptide is not found in the p30 of the N-tropic virus of BALB/c or the XLPN viruses. These results are consistent with the possibility that p30 may determine the N-, B-, or NB-tropism of murine leukemia viruses. In addition, these studies indicate that some of the N-tropic recombinants have experienced recombination within the p30 gene.

Trichinella spiralis: Partial Characterization of Antigens Isolated by Immuno-Affinity Chromatography from the Large-Particle Fraction of the Muscle Larvae

A large-particle fraction, derived from muscle larvae of Trichinella spiralis by a combination of homogenization and differential centrifugation, was treated with 0.1% Triton X-100 and then centrifuged at high speed in order to obtain the soluble portion of this fraction (i.e., S3 fraction). The S3 fraction was then subjected to immuno-affinity chromatography. The antigens were eluted from the column using glycine-HCl buffer (pH 2.5). All 20 antigens were recovered from the S3 fraction in quantities that permitted physical, chemical, and biological determinations to be made on them. Antigens analyzed on SDS-PAGE ranged in molecular weight from 105,000 to 11,000 daltons Their isoelectric points were estimated with slab gel isoelectric focusing and ranged in pI from 4.0 to 9.0. The column-purified antigens were injected into mice together with an equal volume of Freund's complete adjuvant, and 2 wk later all mice were each challenged with 200 muscle larvae given orally. Immunized mice harbored 84% fewer muscle larvae at day 30 postinfection than controls, and were as immune as those mice receiving whole S3 (86% reduction). These results showed that protection-inducing antigens can be separated from the nonprotective portion of the muscle larva by immuno-affinity chromatography without loss of protection-inducing activity.