Two-dimensional Protein Separation Research Articles

High resolution, standard format two-dimensional protein electrophoresis using disposable glass micropipettes and non-dedicated equipment.

A method for high resolution, two-dimensional polyacrylamide gel electrophoresis (PAGE) of proteins on a standard format that utilizes non-dedicated electrophoretic equipment is described. This method combines key features of various previous protocols into an improved approach which can readily be applied by persons experienced only in sodium dodecyl sulfate (SDS)-PAGE. The method yields high-quality two-dimensional protein separations that are reliably reproducible and uniform between different analyses. High protein resolution is attained and preserved primarily by facilitating the execution of the first isoelectric focusing (IEF) dimension and streamlining the initiation of the second (SDS-PAGE) dimension. Important features of this method include the use of piperazine diacrylate cross-linker, disposable glass micropipettes, and conical sample chambers in the IEF dimension. In addition, the equilibration of the first-dimensional gels is accomplished at the same time the gels are loaded onto the second dimension. Loss of protein resolution due to diffusion after the end of the first dimension is reduced by minimizing the time required to load the SDS-PAGE dimension. The method is versatile and adaptable to the analysis of one or many samples. The high degree of resolution, reproducibility and uniformity attained by this method are sufficient to abrogate the need for dedicated equipment in most applications.

Two-dimensional protein separation by microcolumn size-exclusion chromatography-capillary zone electrophoresis

A two-dimensional separation system for the study of proteins consisting of size-exclusion chromatography (SEC) and capillary zone electrophoresis (CZE) is presented. The SEC separation was carried out in a 1 m × 250 μm I.D. microcolumn. The effluent from this SEC microcolumn filled a sample loop on a computer controlled 6-port valve. The contents of this loop were swept past the grounded end of the CZE capillary for electromigration injection. Detection was by on-line UV detection at 214 nm. The system was initially tested with protein test mixtures, then was used to analyze human, horse and bovine sera. Data are presented as three-dimensional chromatoelectropherograms and grayscale images.

A 41-kilodalton protein is a potential substrate for the p210bcr-abl protein-tyrosine kinase in chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is characterized by a translocation involving the c-abl protein-tyrosine kinase gene. A chimeric mRNA is formed containing sequences from a chromosome 22 gene (bcr) at its 5' end and all but the variable exon 1 of c-abl sequence. The product of this mRNA, p210bcr-abl, has constitutively high protein-tyrosine kinase activity. We examined K562 cells and other lines established from CML patients for the presence of phosphotyrosine (P-Tyr)-containing proteins which might be p210bcr-abl substrates. Two-dimensional gel separation of 32P-labeled proteins followed by phosphoamino acid analysis of 25 phosphoproteins, which comprised the major alkali-stable phosphoproteins, indicated that three related proteins of 41 kDa are the most prominent P-Tyr-containing proteins detected by this method. The 41-kDa phosphoproteins are found in two other CML lines that we examined but not in lines of similar lineage isolated from patients with distinct leukemic disease. A protein that comigrates with the major form of pp41 (pp41A) and contains P-Tyr is also found in murine fibroblasts and B-lymphoid cells transformed by Abelson murine leukemia virus, which encodes the v-abl protein, and in platelet-derived growth factor-treated fibroblasts, in which it has been described previously. We analyzed three pairs of Epstein-Barr virus-immortalized B-cell lines from individual CML patients and found that only the lines in which active p210bcr-abl was present contained detectable pp41. We also performed immunoblotting with anti-P-Tyr antibodies on the same CML cell lines and detected at least four other putative substrates of p210bcr-abl, which were undetected with use of the two-dimensional gel technique.

A 41-kilodalton protein is a potential substrate for the p210bcr-abl protein-tyrosine kinase in chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is characterized by a translocation involving the c-abl protein-tyrosine kinase gene. A chimeric mRNA is formed containing sequences from a chromosome 22 gene (bcr) at its 5' end and all but the variable exon 1 of c-abl sequence. The product of this mRNA, p210bcr-abl, has constitutively high protein-tyrosine kinase activity. We examined K562 cells and other lines established from CML patients for the presence of phosphotyrosine (P-Tyr)-containing proteins which might be p210bcr-abl substrates. Two-dimensional gel separation of 32P-labeled proteins followed by phosphoamino acid analysis of 25 phosphoproteins, which comprised the major alkali-stable phosphoproteins, indicated that three related proteins of 41 kDa are the most prominent P-Tyr-containing proteins detected by this method. The 41-kDa phosphoproteins are found in two other CML lines that we examined but not in lines of similar lineage isolated from patients with distinct leukemic disease. A protein that comigrates with the major form of pp41 (pp41A) and contains P-Tyr is also found in murine fibroblasts and B-lymphoid cells transformed by Abelson murine leukemia virus, which encodes the v-abl protein, and in platelet-derived growth factor-treated fibroblasts, in which it has been described previously. We analyzed three pairs of Epstein-Barr virus-immortalized B-cell lines from individual CML patients and found that only the lines in which active p210bcr-abl was present contained detectable pp41. We also performed immunoblotting with anti-P-Tyr antibodies on the same CML cell lines and detected at least four other putative substrates of p210bcr-abl, which were undetected with use of the two-dimensional gel technique.

High- and Small-Molecular-Weight GTP-Binding Proteins in Zymogen Granule Membranes of Rat Pancreatic Acinar Cells

We have detected high- and small-molecular-weight GTP-binding proteins (G- and smg-proteins, respectively) in zymogen granule membranes (ZGM) of rat pancreatic acinar cells. G_i-proteins with molecular weights of 40/41 kDa were detected by pertussis-toxin induced ADP ribosylation and by [α^-32P] GTP photoaffinity labelling in ZGM. Smg-proteins were analysed by [α^-32P] GTP binding, ADP ribosylation with <i>Clostridium botulinum</i> ADP ribosyltransferase C3 and immunoblotting following one- and two-dimensional gel electrophoresis. Two-dimensional separation of ZGM proteins revealed the presence of up to 30 differently charged smg-proteins with molecular masses between 18 and 27 kDa in ZGM. A monoclonal antibody, which recognizes both rab3A and rab3B (clone 42.1), identified 2 proteins with isoelectric points (pI) of 4.89 and 4.96 as proteins closely related to rab3. These proteins were mainly present in ZGM and to a slight extent also in plasma membranes (PM), microsomal membranes (MM) and cytosol. An antibody, which recognizes rab3A only (clone 42.2), did not react with pancreatic proteins. An anti-rap IB antibody recognized a 23 kDa protein localized in the ZGM, PM and to a lesser extent in MM. A monoclonal antibody against all 3 known ras proteins (p21^Ha-ras, p21^ĸi-ras, p21^N-ras) identified ras proteins only in PM and not in ZGM or other membranes. C3-induced ADP ribosylation as indication for the presence of rho and/or rac proteins was detected in PM, MM and cytosol but not in ZGM. The presence of multiple GTP-binding proteins in ZGM suggests a possible role of these proteins in the regulation of exocytosis in pancreatic acinar cells.

Hyaline droplet nephropathy resulting from exposure to 3,5,5-trimethylhexanoyloxybenzene sulfonate

Acute oral dosing of 3,5,5-trimethylhexanoyloxybenzene sulfonate (THBS) to adult male and female rats causes a male rat-specific nephrotoxicity manifested as exacerbation of hyaline droplet formation. This chemical is structurally distinct from the volatile hydrocarbons known to cause male rat-specific kidney lesions. Therefore, to classify THBS as a hyaline droplet-inducing agent, experiments were conducted to determine whether [ 14C]THBS equivalents bound to α2u-globulin and caused the protein to accumulate in male rat kidney cortex. Two-dimensional gel electrophoretic separation of male rat kidney proteins indicated that α2u-globulin levels in kidney increased 24 hr after a single oral dose of THBS (500 mg/kg). Furthermore, a sex-dependent retention of THBS was noted as there was approximately 10 times more THBS equivalent in male rat kidney than in female rat kidney. Equilibrium dialysis experiments indicated that 40% of THBS equivalents bound reversibly to male rat kidney proteins, whereas no interaction between THBS and female rat kidney proteins was detected. Specific binding of THBS to α2u-globulin was determined by anion-exchange HPLC after which metabolites in the α2u-globulin fraction were identified by gas chromatography with parallel radioactivity-mass spectrometry and mass spectrometry-matrix isolation Fourier-transform infrared analysis. Four metabolites of THBS were found in this protein fraction, and the major component (approximately 70%) was identified as the cis ggg-lactone of 3,5,5-trimethylhexanoic acid. Experiments were also conducted in mice to determine whether THBS bound to any mouse kidney proteins, particularly mouse urinary protein. The results indicated that there was no interaction between THBS and mouse urinary protein, a protein which shares siggificant homology with α2u-globulin. These results indicate that THBS treatment exacerbates hyaline droplet formation in male rat kidneys by binding to α2u-globulin, thereby causing the protein to accumulate in the renal cortex. The interaction between THBS and α2u-globulin appears to be unique to this male rat-specific protein as THBS does not interact with a very similar protein found in mice.

Characterisation of human and murine snRNP proteins by two-dimensional gel electrophoresis and phosphopeptide analysis of U1-specific 70K protein variants

The proteins of the major human snRNPs U1, U2, U4/U6 and U5 were characterised by two-dimensional electrophoresis, with isoelectric focussing in the first dimension and SDS-polyacrylamide gel electrophoresis in the second. With the exception of protein F, which exhibits an acidic pl value (pl = 3.3), the snRNP proteins are basic. Post-translational modification was found among the proteins associated specifically with the U1 and U2 particles. The most complex modification pattern was observed for the U1-specific 70K protein. This was found in at least 13 isoelectric variants, with pl values ranging from 6.7 to 8.7; these variants differed also in molecular weight. All of the 70K variants are phosphorylated in the cell. Thin-layer analysis of their tryptic phosphopeptides revealed that the 70K variants have four major phosphopeptides in common, in addition to which at least four additional serine residues are phosphorylated to different extents. The comparative phosphopeptide analysis shows that differential phosphorylation alone is not sufficient to explain the occurrence of the many isoelectric variants of 70K, so that the final charge of the 70K variants is determined both by phosphorylation and by other, as yet unidentified posttranslational modifications. By two-dimensional separation of snRNP proteins obtained from mouse Ehrlich ascites tumour cells, it was shown that the pattern of pl values of the mouse proteins was almost identical with the corresponding pattern for human proteins. Even the complex modification patterns of the 70K protein are identical in mouse and man, indicating that the presence in the cell of so many variants of this protein may have functional importance. The major difference between murine and human snRNP proteins is the absence of protein B' from mouse snRNPs. This suggests that the homologous protein B may be able to carry out the task of protein B'.

The humoral autoimmune response to vasectomy described by immunoblotting from two-dimensional gels and demonstration of a human spermatozoal antigen immunochemically crossreactive with the D2 adhesion molecule

The specificity of human serum antispermatozoal antibodies was analysed by immunoblotting after two-dimensional electrophoretic separation (IEF/PAGE) of human spermatozoal proteins. Spermatozoal proteins with M r 20–200 kDa and p I between 4.5 and 7.8 were analysed for antigenicity. Fifteen sera (four female and 11 male) were analysed for antisperm IgG antibodies. Individual IgG binding patterns were observed. The antisperm antibody response to ligation of the vas deferens was described by immunoblotting analysis of sera taken pre- and post-operatively at regular intervals from three men undergoing vasectomy. The high resolution of spermatozoal antigens allowed a specific description of the humoral response to vasectomy. Both appearance and disappearance of spots were observed in the postoperative period, indicating induction of new antibody producing clones and binding of former free antibodies in circulating immunocomplexes. In one patient five glycosylated proteins induced an IgM response 2 weeks after vasectomy. Four of these autoantigens with M r of 122 kDa, 119 kDa, 104 kDa and 77 kDa and p I of 5.9, 6.1, 7.7 and 6.0, respectively, have previously been shown to be intrinsic membrane proteins (Naaby-Hansen, (1990) J., Reprod. Immunol. 17). An observed immunochemical crossreactivity between the interneuronal adhesion molecule, D2-protein and an antigen extracted from human sperm and teratomas is dicussed.

Epitope mapping of monoclonal antibodies toEscherichia coli ribosomal protein S3

The antigenic structure of Escherichia coli ribosomal protein S3 has been investigated by use of monoclonal antibodies. Six S3-specific monoclonal antibodies secreted by mouse hybridomas have been identified by immunoblotting of two-dimensional ribosomal protein separation gels. By using a competitive enzyme-linked immunosorbent assay, we have divided these monoclonal antibodies into three mutual inhibition groups, members of which are directed to three distinct regions of the S3 molecule. The independence of these monoclonal antibody-defined regions was confirmed by the failure of pairs of monoclonal antibodies from two inhibition groups to block the binding of biotinylated monoclonal antibodies of the third group. To determine the regions recognized by these monoclonal antibodies, chemically cleaved S3 peptides were fractionated by gel filtration and reverse-phase high-performance liquid chromatography. The fractionated peptides were coated on plates and examined for specific interaction with monoclonal antibody by enzyme immunoassay. In this manner, two epitopes have been mapped at the ends of the S3 molecule: one, in the last 22 residues, is recognized by three monoclonal antibodies; and the second, in the first 21 residues, is defined by two monoclonal antibodies. The third S3 epitope, recognized by a single monoclonal antibody, has been localized in a central segment of about 90 residues by gel electrophoresis and immunoblotting. These epitope-mapped monoclonal antibodies are valuable probes for studying S3 structure in situ.

Two‐dimensional electrophoresis of membrane proteins: Separation of myelin proteins

A method for two-dimensional electrophoretic separation of myelin proteins is presented. The first dimension consists of isoelectric focusing of lyophilized and delipidated membrane proteins, solubilized in a mixture of the nonionic detergent Triton X-100, the zwitterionic detergent CHAPS, 9 M urea and carrier ampholytes, and incorporated into a slab gel before separation. Subsequent discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed by moulding the isoelectric focusing slab gel with its supporting glass plate into the stacking gel. This method proved to give highly reproducible results since mechanical forces and thus the risk of stretching, folding or rupture of the isoelectric focusing slab gel is minimized. Furthermore, by immunoblotting, the positions of myelin-associated glycoprotein and 2',3'-cyclic nucleotide 3'-phosphodiesterase were established with specific antibodies.

Growth and soluble proteins of cell cultures derived from explants and protoplasts of Pinus pinaster cotyledons

Pinus pinaster Ait. cell suspension cultures were derived from chopped cotyledons and from cotyledon protoplasts. When transferred after 12 weeks in culture, growth of both cell types showed a lag of 5 days followed by an exponential phase of 14 days for the protoplast-derived cells and 23 days for the organ-derived cells. During the exponential growth phase, packed cell volume of protoplast-derived cultures increased 7-fold and that of organ-derived cultures 13-fold. During the stationary phase, the diameters of protoplast-derived cells averaged 80 microm and those of organ-derived cells 100 microm. After 30 days, the media containing protoplast-derived and organ-derived cells decreased in osmolarity by 50 and 120 mOs per kg water, respectively, and in pH by 1.4 and 2.0 units, respectively. Throughout the growth cycle, protein content per unit of packed cell volume was always at least 35% higher in the protoplast-derived cultures than in the organ-derived cultures. Two-dimensional electrophoretic separation of soluble proteins revealed three peptides present in protoplast-derived cells that were absent from organ-derived cells and two peptides present in organ-derived cells that were absent from protoplast-derived cells. Other peptides differed quantitatively between the cell types.

Application of an affinity electrophoretic and in situ oxidation method to the study of the equine protease inhibitory proteins.

An affinity method was developed to investigate the interaction between protease and protease inhibitor by incorporating a protease incubation step into a two-dimensional electrophoretic separation of the plasma protease inhibitory proteins. This involved the application of the isoelectric focusing gel to filter paper saturated in the protease of choice before being placed on the second-dimensional polyacrylamide electrophoresis gel. General protein staining or immunoblotting was used to detect the protein or ligand in the complex. An in situ oxidation method was developed using the reagent chloramine T to investigate the effect of this reagent on the complexing abilities and inhibitory activities of the protease inhibitory proteins. Oxidation was performed either after electrophoresis prior to staining for enzyme inhibition or during two-dimensional electrophoresis prior to the aforementioned protease incubation. The latter allowed the effect of oxidation on complex formation to be examined. Whole plasmas were utilized as the sources of protease inhibitory proteins with the human and mouse being used as models. The equine protease inhibitory system was examined by the two methods and shown to consist of three classes of inhibitory proteins based on their susceptibilities to oxidation and abilities to form complexes with various proteases.

Detection of spatially- and stage-specific proteins in extracts from single embryos of the domesticated carrot

Abstract Single embryos, representing each of four distinct morphological stages, were selected from cultures of the domesticated carrot for analysis of total [3SS]methionine-labelled proteins. Following exposure to radiolabel for 12 to 18 h, embryos were individually disrupted in a 3 mm diameter, precisely-matched, plastic mortar and pestle. Radiolabelled proteins extracted by this procedure were separated by two-dimensional electrophoresis procedures, consisting of isoelectric focusing in 1 mm tubes, followed by SDS-PAGE in a small slab gel. Comparisons of autoradiographs of these gels revealed that the levels of a number of proteins were modulated during the conversion of disordered callus cells into maturing embryos. In addition, miniature surgical techniques were used to separate the apex (cotyledon end) from the base (root end) of late-stage embryos, for extraction of proteins and analysis of spatial differences in protein distribution. About five proteins in extracts from each section were observed to be synthesized at different rates in the two halves, indicating that there are molecular correlates for early polarized growth. About half of the proteins, whose appearances were unique to apical and basal sections of embryos, were also observed to fluctuate in comparisons of autoradiographs of two-dimensional protein separations from embryos at different developmental stages.

Identification of mouse brain proteins after two-dimensional electrophoresis and electroblotting by microsequence analysis and amino acid composition analysis.

Two-dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures. This method avoids the drawbacks of classical purification and isolation methods which involve time-consuming operations with low resolution and, often, insufficient yields. Excellent overall yields of minor amounts (in the low microgram range) using this method allow for sequence determination of yet inaccessible proteins. Solubilized cell proteins of mouse brain were separated by high resolution two-dimensional electrophoresis and electroblotted onto a siliconized glass fiber membrane. The immobilized proteins were stained with Coomassie Brilliant Blue R-250, and twelve proteins spots were then submitted to both Edman degradation and amino acid analysis. Proteins were identified by comparison of the experimentally determined amino acid composition with a dataset derived from the Protein Identification Resource (PIR) protein sequence database. Eight out of twelve proteins tested were identified by amino acid analysis and confirmed by N-terminal sequence determination.

Alterations in the phenotype of plant cells studied by NH 2 -terminal amino acid-sequence analysis of proteins electroblotted from two-dimensional gel-separated total extracts

Phenotypic alterations induced by the cytokinin 6-benzylaminopurine in cell suspensions of Nicotiana plumbaginifolia were studied at the level of the NH(2)-terminal sequence of the constituent proteins. Total protein extracts were separated by classical two-dimensional PAGE, and the proteins were recovered by electroblotting onto support materials allowing direct gas-phase sequence analysis of the immobilized proteins. The systems used consist of an efficient electrotransfer buffer (50 mM Tris borate, pH 8.3) in combination with either glass-fiber sheets to which poly(4-vinyl-N-methylpyridinium iodide) is adsorbed or with membranes of polyvinylidene difluoride. The former is an improved version of our previously reported Polybrene-coated glass-fiber sheets and was found to be at least twice as efficient as the polyvinylidene difluoride blots. Thirteen proteins were selected for analysis. They were either induced, repressed, or independent of cytokinin. Ten proteins yielded a sequence, ranging from 10 to 38 residues. Three of the studied Nicotiana proteins show a degree of homology higher than 85% with the amino acid sequences of other eukaryotic proteins-triose-phosphate isomerase, Mn superoxide dismutase, and (1,3)-beta-glucanase. The latter enzyme was repressed by the plant hormone. This study demonstrates that proteins associated with phenotypic variations in cells can now be sequenced by a straightforward procedure involving two-dimensional gel separation of total cellular proteins, recovery by electroblotting, and gas-phase sequence analysis of the immobilized proteins.

Identity of the proliferating cell nuclear antigen and cyclin.

Studies of growth regulation and cellular transformation will be assisted by the identification of proteins that are preferentially synthesized in dividing cells. The 'proliferating cell nuclear antigen' ( PCNA ), distinguished by its apparent association with cell division, is defined by reaction with an antibody found in the autoimmune disease systemic lupus erythematosus (SLE). This antibody reacts with proliferating cells including tumour cells but gives weak or undetectable immunofluorescence with resting cells of normal tissues. Peripheral blood lymphocytes are devoid of PCNA until activated by mitogen in vitro. In synchronized cultures its level and distribution fluctuate through the cell cycle, with a striking accumulation in the nucleolus late in the G1 phase and early in the S phase. Many of these properties are shared by ' cyclin '. This nuclear protein, identified by its position in a two-dimensional separation of cell proteins, is also transformation-sensitive and is preferentially synthesized in the S phase. We establish here that PCNA and cyclin are identical, and show that PCNA is an acidic nuclear protein of apparent molecular weight 35,000.

Tissue specificity of porcine zona pellucida antigens in pigs, monkeys and humans

The specificity of human serum antispermatozoal antibodies was analysed by immunoblotting after two-dimensional electrophoretic separation (IEF/PAGE) of human spermatozoal proteins. Spermatozoal proteins with Mr 20–200 kDa and pI between 4.5 and 7.8 were analysed for antigenicity. Fifteen sera (four female and 11 male) were analysed for antisperm IgG antibodies. Individual IgG binding patterns were observed. The antisperm antibody response to ligation of the vas deferens was described by immunoblotting analysis of sera taken pre- and post-operatively at regular intervals from three men undergoing vasectomy. The high resolution of spermatozoal antigens allowed a specific description of the humoral response to vasectomy. Both appearance and disappearance of spots were observed in the postoperative period, indicating induction of new antibody producing clones and binding of former free antibodies in circulating immunocomplexes. In one patient five glycosylated proteins induced an IgM response 2 weeks after vasectomy. Four of these autoantigens with Mr of 122 kDa, 119 kDa, 104 kDa and 77 kDa and pI of 5.9, 6.1, 7.7 and 6.0, respectively, have previously been shown to be intrinsic membrane proteins (Naaby-Hansen, (1990) J., Reprod. Immunol. 17). An observed immunochemical crossreactivity between the interneuronal adhesion molecule, D2-protein and an antigen extracted from human sperm and teratomas is dicussed.

ELISA : Determination and titration of antisperm antibodies

The specificity of human serum antispermatozoal antibodies was analysed by immunoblotting after two-dimensional electrophoretic separation (IEF/PAGE) of human spermatozoal proteins. Spermatozoal proteins with Mr 20–200 kDa and pI between 4.5 and 7.8 were analysed for antigenicity. Fifteen sera (four female and 11 male) were analysed for antisperm IgG antibodies. Individual IgG binding patterns were observed. The antisperm antibody response to ligation of the vas deferens was described by immunoblotting analysis of sera taken pre- and post-operatively at regular intervals from three men undergoing vasectomy. The high resolution of spermatozoal antigens allowed a specific description of the humoral response to vasectomy. Both appearance and disappearance of spots were observed in the postoperative period, indicating induction of new antibody producing clones and binding of former free antibodies in circulating immunocomplexes. In one patient five glycosylated proteins induced an IgM response 2 weeks after vasectomy. Four of these autoantigens with Mr of 122 kDa, 119 kDa, 104 kDa and 77 kDa and pI of 5.9, 6.1, 7.7 and 6.0, respectively, have previously been shown to be intrinsic membrane proteins (Naaby-Hansen, (1990) J., Reprod. Immunol. 17). An observed immunochemical crossreactivity between the interneuronal adhesion molecule, D2-protein and an antigen extracted from human sperm and teratomas is dicussed.

Blue light inhibits slime mold differentiation at the mRNA level.

The influence of blue light on protein synthesis in spherulating Physarum polycephalum microplasmodia was studied using two-dimensional protein separation techniques. The starvation-induced plasmodium-spherule transition proceeds in the dark and is accompanied by the synthesis of 20 major differentiation-specific proteins as revealed by in vivo labelling with [35S]methionine. Three of these proteins are identical with cell wall components with respect to their mol. wts. (35 K, 34 K and 14 K) and isoelectric points. Spherulation is also accompanied by the appearance of 26 prominent differentiation-specific mRNA species translatable in the rabbit reticulocyte cell-free system. Six of the proteins synthesized in vitro co-migrate on two-dimensional gels with proteins labelled in vivo, two of them being cell wall components. Blue light, which inhibits spherulation completely, inhibits also the synthesis of spherule proteins and of spherule-specific mRNA activity. Only three protein components are induced by blue light, indicating that illumination does not induce a novel differentiated plasmodial state.

15] Very-high-resolution two-dimensional electrophoretic separation of proteins on giant gels

15] Very-high-resolution two-dimensional electrophoretic separation of proteins on giant gels