One-dimensional Polyacrylamide Gel Electrophoresis Research Articles

Isolation of lumenal proteins from spinach thylakoid membranes by Triton X-114 phase partitioning

The proteins present in the thylakoid lumen of higher plant chloroplasts have not been rigorously examined. In this communication we present a simple and rapid procedure for the isolation of the soluble proteins and extrinsic membrane proteins present in the thylakoid lumen from spinach. Our procedure involves extensive washing of the thylakoid membranes followed by Triton X-114 phase partitioning. When analyzed by one-dimensional polyacrylamide gel electrophoresis (PAGE), we obtain results which are very similar to those obtained by Kieselbach et al. using more classical methods [T. Kieselbach, A. Hagman, B. Andersson, W.P. Schroder, J. Biol. Chem. 273 (1998) 6710–6716]. About 25 major proteins are observed upon Coomassie blue staining. Upon two-dimensional isoelectric focusing–sodium dodecyl sulfate–PAGE and either Coomassie blue or silver staining, however, numerous other protein components are resolved. Our findings indicate that the total number of proteins (soluble and extrinsic membrane) present in the lumen may exceed 150.

Glutamine synthetase in experimental meningitis: increased ratio of the subunits 3 and 2 may indicate enhanced activity

Glutamine synthetase (GS) activity is higher in the neocortex but not in the hippocampal formation of rabbit brain during Streptococcus pneumoniae meningitis compared to the respective brain region of uninfected control animals. One-dimensional polyacrylamide gel electrophoresis (1D-SDS-PAGE) revealed an apparent molecular mass ( M r) of 44 000 Dalton (Da) for GS from rabbit brain. After two-dimensional gel electrophoresis (2D-PAGE), followed by Coomassie-blue staining, GS separated into three distinct spots (S1, S2, S3). One additional spot (S4) occurred on the immunoblot. All four GS spots exhibited the same M r (44 000 Da), but differed in their isoelectric points. Densitometric evaluation of the two-dimensional maps revealed a strong increase of optical density (OD) of S3 in the frontal cortex of infected animals. The calculated OD ratio S3/S2 in the frontal cortex from rabbits with meningitis was 1.75±0.68 (mean±standard deviation). Compared to controls (0.85±0.39), this value was significantly increased ( p=0.0006). In the hippocampal formation, the ratio S3/S2 was nearly unchanged during meningitis. It is suggested that the ratio S3/S2 may indicate a neuroprotective feature of rabbit brain during meningitis since neuronal apoptosis occurs only in the dentate gyrus and not in the frontal cortex.

Identification of proteins from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis using electrospray quadrupole-time-of-flight tandem mass spectrometry.

The potential of electrospray tandem mass spectrometry using a quadrupole-time-of-flight tandem mass spectrometer was evaluated for the identification of two unknown proteins from one-dimensional polyacrylamide gel electrophoresis (1-D-PAGE). Two proteins from cellular cultures of mammary epithelia were purified by 1D-PAGE. Their identification was achieved using peptide sequence tags generated by LC/Q-TOF-MS/MS, whereas MALDI-TOF mass mapping failed for these proteins obtained from simple 1D-PAGE separation.

Use of Isoenzymes to Differentiate Growth Categories of Pericopsis Mooniana Trees

Leaf isoenzymes of Pericopsis mooniana from twenty-one trees at forest plantation were evaluated for their use in identification of elite trees among heterogeneous population. Trees were grouped morphologically, before leaf extracts were separated by one-dimensional polyacrylamide gel electrophoresis. Isoenzyme analysis were carried out for peroxidase, esterase, alcohol dehydrogenase, formate dehydrogenase, acid phosphatase, aspartate aminotransferase, isocitrate dehydrogenase, malate dehydrogenase, leucine aminopeptidase, phosphoglucoisomerase, phosphogluconate dehydrogenase, phosphoglucomutase, and shikimate dehydrogenase. From the thirteen enzymes studied only four gave distinct banding patterns. Level of significance of appearing particular band for each enzyme of a given category was investigated using χ2-test, followed by cluster analysis for categorization. The isozyme type A of formate dehydrogenase showed promising results that could be used for differentiating trees of categories investigated.

Identification by In Vitro Translation and Northern Blot Analysis of Heat Shock mRNAs Isolated from Wheat Seeds Exposed to Different Temperatures During Ripening

Poly(A)+RNA isolated from durum and common wheat seeds exposed to different thermal regimes during ripening, was translated in vitro using a rabbit reticulocyte system. The modification of protein synthesis was studied with particular regard to the heat shock proteins produced under high temperature conditions. One-dimensional polyacrylamide gel electrophoresis analysis showed products ranging in size from 14 to 100 kDa, some of which were present only when mRNA samples from high temperature-treated plants were translated. The mRNAs were also analysed by Northern hybridization with specific probes for heat shock proteins. The results clearly show that wheat plants respond to thermal stress by triggering the typical mechanisms of the heat shock response including activation of the heat shock genes, in developing grains as well as other plant parts.

Effect of recombinant human megakaryocyte growth and development factor coupled with polyethylene glycol on the platelet storage lesion.

Platelet production is regulated by a thrombopoietic growth factor (Mpl ligand). The receptor for this platelet growth factor (Mpl) is expressed on the platelet surface membrane. A recombinant thrombopoietic cytokine, recombinant human megakaryocyte growth and development factor coupled with polyethylene glycol (PEG-rHuMGDF), was added to apheresis platelets in vitro to determine whether Mpl ligand-receptor binding produced any beneficial or adverse effect on the development of the platelet storage lesion during 5 days of storage. This study was designed as a dose-response protocol to determine the effects of adding increasing concentrations of PEG-rHuMGDF (0.0 [control], 2.5, 25, and 250 ng/mL) to apheresis platelets stored in two types of plastic storage containers. The increasing concentrations of PEG-rHuMGDF used simulated the theoretical peak plasma level attained in vivo, with an intravenous dose of 0, 0.1, 1.0 and 10 microg per kg of PEG-rHuMGDF. The platelets were stored with agitation at 20 to 24 degrees C for 5 days. A battery of in vitro assays was performed on storage Days 1 and 5, including pH, blood gases, platelet count, lactate dehydrogenase, mean platelet volume, glucose, lactate, osmotic recovery, morphology score, CD62P, and one-dimensional polyacrylamide gel electrophoresis analyses. Analysis of results on both Day 1 and Day 5 showed no significant differences among any of the three PEG-rHuMGDF doses and the control group, for any in vitro assay. One-dimensional polyacrylamide gel electrophoresis showed no changes among the platelet protein patterns for the three PEG-rHuMGDF doses studied or the control. Storage-induced changes, however, did occur equally in all four groups of platelets over the 5 days of storage. The addition to stored apheresis platelets of up to 10 microg per kg of PEG-rHuMGDF (250 ng/mL), followed by 5 days of storage at standard conditions, does not appear to promote or retard development of the platelet storage lesion.

Effect of mild acid treatment on the survival, enteropathogenicity, and protein production in vibrio parahaemolyticus.

Vibrio parahaemolyticus is an important food-borne enteropathogen that encounters various adverse conditions in its native environment or during infection. Effects of mild acid treatment on survival under stress conditions, enteropathogenicity, and protein production in this pathogen were investigated. Logarithmically grown cells, at pH 7.5 shifted to pH 5.0 for 30 min, were more resistant to subsequent acid challenge at pH 4.4. A two-phase adaptive procedure (pH 5.8 for 30 min; pH 5.0 for 30 min) was better than a single-phase procedure for enhancing the acid tolerance of this pathogen. The acid-adapted cells were cross-protected against the challenges of low salinity and thermal inactivation. One-dimensional polyacrylamide gel electrophoresis revealed that proteins with molecular masses of 6.4, 9.0, 13.6, 16.3, 18.9, 22.9, 24.4, 28.3, 33. 9, 36.9, 41.2, 47.6, 58.1, 65.6, 80.5, 88.2, and 96.9 kDa were induced or significantly enhanced, while proteins of 25.3, 30.1, 30. 7, and 91.7 kDa were significantly inhibited. Two-dimensional polyacrylamide gel electrophoresis revealed that 20 species of proteins were induced or significantly enhanced, while 26 species were inhibited. In assays conducted using the suckling mouse model, enteropathogenicity of the acid-adapted cells was significantly enhanced in terms of intestine/body weight ratio and in vivo recovery of infected cells.

Acute changes in urine protein excretion may predict chronic ifosfamide nephrotoxicity: a preliminary observation.

To evaluate proteinuria occurring early after ifosfamide therapy and to assess the use of changes in proteinuria in the prediction of severe chronic nephrotoxicity. One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis was used to characterize urine protein excretion in 12 children with solid tumours before and after the first course of ifosfamide treatment, and in 24 healthy children. Chronic nephrotoxicity was evaluated at 6 months after ifosfamide treatment and graded as none, mild, moderate or severe. Urine from healthy children and from 10 of 12 patients before ifosfamide therapy showed a protein band with a molecular weight (95.4 kDa) corresponding to that of Tamm-Horsfall protein but no lower molecular weight proteins. After the first course of ifosfamide this 95.4-kDa protein was lost in six of ten patients with a concomitant appearance of a low molecular weight proteinuria (< 70 kDa) in eight. Tamm-Horsfall protein was lost in two of five patients who subsequently developed no or mild nephrotoxicity and in four of five patients who subsequently developed moderate or severe nephrotoxicity. Early subclinical changes in urine protein excretion after ifosfamide, manifested by a loss of Tamm-Horsfall protein excretion, may be predictive of subsequent chronic nephrotoxicity.

Stored Renin Isoforms in the Developing Rat Kidney

Fetal rat kidney contains renin in renal microvasculature, whereas adult rat kidney contains renin predominantly in juxtaglomerular cells. It is hypothesized that renin isoforms stored within these renal tissues may differ chemically and functionally. To test this hypothesis, stored renin isoforms in fetal and adult rat kidney were compared by isolating renin from adult and fetal kidney homogenate with pepstatin agarose. Pepstatin-eluted renin isoforms were separated by relative molecular size using one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE), or by isoelectric point (pI) and size using two-dimensional (2D) gel electrophoresis. Isoforms were identified either by silver staining or immunoblotting. One-dimensional polyacrylamide gel electrophoresis of pepstatin-treated kidney homogenates showed a silver-stained band in the range of ∼45 kDa, which corresponded to a silver-stained spot consistently seen on 2D gels. In fetal kidney homogenate, the ∼45 kDa band had a pI of 5.3 ± 0.1, whereas the corresponding band in adult samples had a basic pI of 6.0 ± 0.05. Angiotensin I generation was measured to assess renin enzymatic activity. There was significantly more inactive renin in fetal kidney homogenate than in adult kidney homogenate (60.2 ± 22.4 v 9.6 ± 4.0 ng AI/mg protein/h, P < .05). There was significantly less active renin in fetal kidney homogenate than in adult kidney homogenate (5.4 ± 0.4 v 36.5 ± 14.2 ng AI/mg protein/h, P < .05). The average total renin activity in fetal kidney homogenate was significantly higher than in adult kidney homogenate (65.6 ± 22.3 v 46.0 ± 15.2, P < .05). These results demonstrate major differences in the physical and enzymatic forms of stored renin found in fetal and adult kidney. It is speculated that these variations in stored renin isoforms play a role in the developmental differential regulation of the intrarenal renin angiotensin system.

Increases in fragmented glial fibrillary acidic protein levels in the spinal cords of patients with amyotrophic lateral sclerosis.

Using one-dimensional polyacrylamide gel electrophoresis, we analyzed protein fractions extracted from the spinal cords of patients with amyotrophic lateral sclerosis (ALS). Several protein bands with molecular weights of 35-55 kDa were stained with Coomassie brilliant blue much more intensely in the ALS than in the non-ALS spinal cord. Glial fibrillary acidic protein (GFAP) immunoreactivity showed a significant decrease of 50 and 45 kDa band and increase in fragmented 36 and 37 kDa bands, which represented GFAP fragments devoid of 59 and 40 residues from the N-terminal, respectively, as determined by protein sequence analysis. Immunohistochemical examination of ALS spinal cord transections demonstrated increased GFAP-stained astrocytes in the shrunken ventral horn with massive degeneration of motoneurons. These results will provide new insight into the possible role of astrocytes in the pathophysiology and/or pathogenesis of ALS.

Decreased Retinoylation in NIH 3T3 Cells Transformed with Activated Ha-ras

Retinoylation (retinoic acid acylation) is a posttranslation modification of proteins occurring in a variety of mammalian cell lines andin vivo.To gain further knowledge of the role of retinoylation we studied it in NIH 3T3 cells and NIH 3T3 cells transformed by an activated Ha-rasoncogene (NIH Ha-ras-3T3 cells). In serum-free medium retinoic acid (RA) inhibited growth of NIH 3T3 cells but did not inhibit growth of NIH Ha-ras-3T3 cells. After incubation with [3H]RA, the level of retinoylated protein in NIH 3T3 cells was about 1.5-fold greater than in NIH Ha-ras-3T3 cells. On one-dimensional polyacrylamide gel electrophoresis, both the rate and the extent of retinoylation were greater in NIH 3T3 cells. We detected about 40 retinoylated proteins in NIH 3T3 cells by two-dimensional polyacrylamide gel electrophoresis. Only about 15 proteins were retinoylated, but at reduced levels, in NIH Ha-ras-3T3 cells. These results suggest that the activatedrasoncogene inhibits retinoylation. This inhibition may in turn be related to the loss of other RA responses of NIH 3T3 cells, including growth inhibition, retinoic acid catabolism, down-regulation of fibronectin biosynthesis, and induction of tissue-type transglutaminase, which are not seen to the same extent in NIH Ha-ras-3T3 cells.

Effect of enucleation on protein synthesis during maturation of bovine oocytes in vitro.

The role of the nucleus in protein synthesis reprogramming during oocyte maturation was examined in immature or mature bovine oocytes, enucleated at the germinal vesicle (GV) stage or the metaphase II (MII) stage. Cumulus-oocyte complexes (COCs) were denuded before or after maturation in vitro. Denuded oocytes were (i) enucleated at the GV or MII stage (after DNA staining and ultraviolet (UV) exposure), (ii) stained and exposed to UV but not enucleated, or (iii) used as controls. After treatment, oocytes were labelled for 4 h with 35S-methionine or were matured for 24 h before labelling. GV- or MII- karyoplasts and small portions of cytoplasm (cytoplasts), removed during enucleation, were also labelled. Labelled oocytes, karyoplasts or cytoplasts were prepared for one-dimensional polyacrylamide gel electrophoresis. Incorporation of labelled methionine into oocyte protein was measured. Enucleation did not affect protein synthesis reprogramming, but incorporation of 35S-methionine in immature UV-stained oocytes was high--possibly due to nuclear repair mechanisms. Protein profiles of GV- and MII- karyoplasts differed from those of immature and mature oocytes. In conclusion, normal protein synthesis reprogramming in the cytoplasm can occur in the absence of the nucleus, and specific proteins are synthesized in the nuclear region.

Plasma protease inhibitor (PI) system in the laboratory opossum, Monodelphis domestica.

Protease inhibitor (PI) polymorphism was observed in the laboratory opossum, Monodelphis domestica, by either one-dimensional acid polyacrylamide gel electrophoresis (PAGE; pH 4.6) or isoelectric focusing (pH 3.5-5.0) followed by immunoblotting with rabbit antiserum to human alpha 1-antitrypsin; but acid PAGE produced superior resolution of the PI proteins. Family studies demonstrated an inheritance of nine codominant autosomal alleles, PID, PIE, PIF, PIG, PIH, PII, PIJ, PIK, and PIM, and a population study revealed frequencies of 0.411, 0.010, 0.341, 0.034, 0.023, 0.071, 0.035, 0.020, and 0.055, respectively.

Molecular characterization of cellular proteins from colorectal tumors.

Recent evidence has suggested that progressive stages of colorectal tumorigenesis can be defined by a sequence of genetic events characterized by altered expression of certain genes and the appearance of cancer-specific proteins. Although the significance of these events is still not clear, expression of cancer-specific protein components may be directly involved in the neoplastic transformation. The purpose of the present study was to compare molecular characteristics of cellular proteins from human colorectal tumors and normal colonic mucosa. Normal mucosa and colorectal tumors from 18 patients were fractionated by a differential centrifugation scheme into four cellular fractions, i.e., nuclear, mitochondrial (10P), microsomal (100P) and cytosolic (100S). The proteins of these fractions from normal and tumorigenic mucosa were analyzed by one-dimensional polyacrylamide gel electrophoresis followed by Coomassie brilliant blue R-250 and silver nitrate staining. Nuclear proteins from normal and neoplastic tissues which had revealed the most significant diversities were further characterized by two-dimensional gel electrophoresis. Electrophoretically cancer-specific nuclear proteins in the molecular mass zone 35-40 kDa were used as immunogen to produce rabbit polyclonal antibodies. Electrophoretic analysis by one-dimensional gel electrophoresis showed clear differences in molecular characteristics of cellular proteins between normal and tumorigenic mucosa, especially among nuclear fractions. The latter were also confirmed by their two-dimensional electrophoresis results. Rabbit antibodies raised against electrophoretically specific nuclear proteins characterized by molecular mass of 35-40 kDa cross-reacted with 36 kDa polypeptide in 15 of 18 (83.3%) studied nuclear fractions of colorectal tumors but not with any normal mucosa. In some cases, nuclear cancer-associated components of 38 and 40 kDa were also recognized by these antibodies. During colorectal carcinogenesis, specific expression of several nuclear proteins takes place. One of them, the polypeptide of 36 kDa not found in normal colonic epithelium, was shared by over 83% of the studied carcinomas despite variations in detailed cancer properties. This particular nuclear protein may be considered as a potential marker for the colon malignancy.

Heterogeneity of human intestinal spirochaetes demonstrated by one-dimensional polyacrylamide gel electrophoresis of proteins visualised by 35S-methionine labelling and Coomassie Blue staining

The relatedness of strains of a human intestinal spirochaete was investigated by comparison of electrophoretic protein profiles produced by Coomassie Blue staining of proteins separated by polyacrylamide gel electrophoresis (PAGE) of lysed organisms and by examination of autoradiographs following PAGE of lysed (35)S-methionine-labelled organisms. A wide diversity of strains was revealed by both techniques but clustering of strains was different by the two methods. These findings support the view that the human intestinal spirochaetes comprise a group of bacteria of considerable heterogeneity.

Genetic control of PI and GC variants in the American mink.

Genetic polymorphism of the serum alpha-protease inhibitor (PI) and group-specific component (GC) in minks was revealed using one-dimensional polyacrylamide gel electrophoresis and immunoblotting. Two codominant alleles were identified at each of the two loci. The data ruled out the possibility of any linkage between the PI, GC and the coat colour gene Crystal (Cr).

Protein profiles of Streptomyces aureofaciens producing tetracyclines: Reappraisal of the effect of benzyl thiocyanate

Cell protein profiles of submerged cultures of Streptomyces aureofaciens cultivated in the absence or presence of 12 microM benzyl thiocyanate (BT) were analyzed by one-dimensional SDS polyacrylamide gel electrophoresis. Substantial increase in the intensity of the 13, 35, 37, 60, and 100 kDa protein bands was observed in cultures treated with BT. Similar increase in the 35, 37, and 60 kDa bands was found in a mutant blocked in the last chlortetracycline biosynthesis step. Effect of BT on the solid medium-grown cultures was also observed, with a more intensive substrate mycelium pigmentation and alteration in the spore size and shape as the most characteristic features. Earlier studies of BT effect involving those on the stimulation of chlortetracycline biosynthesis are summarized and a possible signal-transducing mechanism is discussed from the point of view of adaptation of S. aureofaciens to the uncoupling of oxidative phosphorylation.

Protein synthesis and degradation in flight muscles of adult crickets (Gryllus bimaculatus)

The development and degeneration of the flight muscles in adult crickets, Gryllus bimaculatus, were studied (1) by determination of the total protein content, (2) by SDS one-dimensional polyacrylamide gel electrophoresis (SDS&shy;PAGE) of muscle protein and (3) by in vitro culturing of the muscle. The total protein content of the dorso-longitudinal muscle (DLM) and metathoracic dorso-ventral muscle (DVM) increased during the early days of adult life in both sexes. This high protein content was maintained for at least a further 10 days in some individuals, while in others it declined to a low level. Mesothoracic DVMs in males also showed an increase in protein content after adult emergence but did not undergo histolysis, whereas those in females showed no significant temporal change in protein content. Removal of hind wings or artificial de-alation was found to be useful in inducing degeneration of DLMs and metathoracic DVMs. This treatment also stimulated ovarian development in females. An analysis by SDS&shy;PAGE provided no evidence for new protein synthesis prior to or during flight muscle degeneration. A high rate of [3H]- or [35S]methionine incorporation was observed in DLMs taken from newly emerged adults, but, in intact crickets, the rate declined rapidly during the first 3 days of adult life, a pattern consistent with that obtained from the measurement of total protein content. Compared with DLMs removed from intact crickets, DLMs taken from de-alated crickets showed reduced rates of protein synthesis during in vitro culturing. This, together with the onset of protein degradation, appears to cause the rapid decrease in total protein content of the muscle in de-alated crickets.

Activation of the g protein in porcine oocytes

Fertilization in most animal species involves a transient increase in the intracellular free Ca” concentration of the oocyte in the form of a CaPt wave starting from the site of sperm attachment. It is thought that the origin of this Ca” is inositol triphosphate (IP,) induced Ca” release from the endoplasmic reticulum. IP, is derived from the hydrolyses of phosphatidylinositol4,5biphosphate (PIP,) which is catalyzed by the enzyme phospholipase C under regulation of a G protein. Thus the G protein mediated PIP, hydrolysis seems to play an important role in oocyte activation. G proteins or guanine nucleotide binding proteins are a class of membrane proteins that couple membrane receptors to membrane effector enzymes. They are present in a variety of cells, and in oocytes their activation by GTP-7-S (a hydrolysisresistant analog of GTP) causes resumption of meiosis in many species. The present study was undertaken to determine the possibility of porcine oocyte activation through a G protein. Oocytes from slaughterhouse-obtained porcine ovaries were matured in Waymouth medium supplemented with 2.5 mM pyruvate, 10 IU/ml PMSG, 10 IU/ml hCG, 10% (v/v) heat-treated fetal calf serum (Sigma Chemical Co., St. Louis, MO) and 10% (v/v) porcine follicular fluid for 20 h, then in the same medium without hormonal supplement for an additional 16 h. Mature oocytes were stripped of their cumulus cells by vortexing in 0.3 M mannitol solution containing 0.3 mg/ml hyaluronidase. Then 40 pl of 1 mM GTP-7-S (Sigma Chemical Co., St. Louis, MO) was injected near the center of the oocytes using a microinjector (Narishige Co. Ltd, Tokyo, Japan). The carrier medium in which GTP-7-S was dissolved consisted of 100 PM EGTA and 10 mM Hepes buffered at pH 7.0. Control oocytes were injected with either 40 pl of 1 mM GDP-p-S, a metabolically stable analog of GDP (which inactivates G proteins) or carrier medium. Injected oocytes were incubated in Hepes-buffered Tyrodes medium (HbT) for six hours then they were evaluated for some of the early and late events of oocyte activation. Transmission electron microscopy revealed that in contrast to GDP-p-S, GTP-rS caused the release of the vast majority of the cortical granules. It also triggered changes in the protein profiles (i.e., dephosphorylation of a 25 kDa protein) as determined by one-dimensional polyacrylamide gel electrophoresis (1D SDS PAGE). Following GTP-7-S injection 71.4% (190/266) of the mature oocytes formed pronuclei, which was significantly higher (P<O.OOl) than the rates of pronuclear formation in oocytes injected with GDP-p-S (23.7%; 67/282) or carrier medium (25.9%; 52/201). Furthermore, following 7 d of incubation in ligated porcine oviducts 13.1 ok ( 10/76) of the transferred injected oocytes developed to the compact morula/blastocyst stage. We conclude that a signal transducing pathway involving a G protein does exist in the porcine oocyte, and by stimulating the G protein with GTPq-S porcine oocytes can readily be activated.

Chapter 15 Electrophoretic Separation of Dynein Heavy Chains

Publisher Summary Molecular studies of axonemal dyneins have demonstrated that each outer and inner dynein arm contains at least two multiple dynein heavy chains (DHCs) and that inner dynein arms may be composed of different heavy chains along the length of the axoneme. It is probable that each kind of motile axoneme contains multiple DHCs. All DHCs characterized until now cannot be resolved by two-dimensional poly-acrylamide gel electrophoresis and are resolved by one-dimensional polyacrylamide gel electrophoresis only under specific conditions. The electrophoretic procedure presented in this chapter is a modified version of the procedure of Neville. It can be applied to resolve at least some of the DHCs present in cilia or eukaryotic flagella. It was developed to obtain optimal resolution of the 9 or 11 DHCs that are assembled in the axoneme of Chlamydomonas flagella. The procedure is capable of resolving six bands, each comprising one or two or three of the DHCs. Although changes in the composition of the gel or buffers used for the electrophoresis cause changes in the electrophoretic patterns formed by the DHCs, all DHCs cannot be separated by gel electrophoresis alone because they are so similar in structure and present in the axonemes at different concentrations. Complete separation of DHC subsets can be achieved through gel electrophoresis if the number of DHCs under analysis is reduced by mutation or chromatographic fractionation. The chapter discusses the chemicals involved, the solutions, and the apparatus needed. Nine stock solutions are prepared and kept at room temperature. The electrophoresis is performed in a homemade, vertical apparatus that holds 35.5-cm-wide, 26.5-cm-long, and 0.4-cm-thick glass plates. Thinner glass plates are not adequate for optimal resolution of dynein heavy chains because they are flexible and do not provide a gel slab that has homogeneous thickness. Polymerizations are performed at room temperatures.