Bands In Agarose Gel Electrophoresis Research Articles

Proper application of DNA dyes in agarose gel electrophoresis.

Various dyes are used to visualize DNA bands in agarose gel electrophoresis (AGE) by the methods of pre- or post-staining. The DNA dye user's guides generally state that the binding of the dye to DNA will affect DNA mobility in electrophoresis, thus recommending post-staining for accurate measurement of DNA size. However, many AGE performers prefer pre-staining procedures for reasons such as convenience, real-time observation of DNA bands, and/or the use of a minimal amount of dye. The detrimental effect of the dye on DNA mobility and the associated risk for inaccurate measurement of DNA size are often overlooked by AGE performers. Here we quantitatively determine the impact on DNA migration imposed by frequently used dyes, including GelRed, ethidium bromide (EB), and Gold View. It was observed that pre-staining with GelRed and EB significantly slowed down DNA migration to cause as much as 39.1% overestimation on the size of sample DNA, whereas Gold View had little effect. The slowdown of DNA migration increased with dye concentration until it plateaued when the dye concentration reached a saturated level. Thus, to take advantage of pre-staining, saturated levels of DNA dyes should always be applied for both DNA samples and DNA markers to ensure a fair comparison of DNA sizes. In addition, GelRed and EB display much higher sensitivity than Gold View in the detection of DNA bands in post-staining. The saturated concentrations, cost considerations, and other useful features of these frequently used dyes are summarized for the information of AGE performers.

Development and evaluation of a high-sensitivity RT-PCR lateral flow assay for early detection of HIV-1 infection

Early diagnosis of HIV-1 is crucial to minimize transmission, morbidity, and mortality, particularly for neonates with developing immune systems. This study aimed to develop and evaluate a simplified, high-sensitivity assay for early HIV-1 detection before seroconversion. The assay utilizes reverse-transcription-polymerase chain reaction (RT-PCR) to amplify the HIV-1 RNA protease gene. Digoxigenin (dig)-labeled forward, and biotin-labeled universal reverse primers are used, generating digoxigenin-amplicon-biotin (DAB) products. These products are detected using a lateral flow assay (LFA) containing a conjugated pad with colloidal gold-labeled 6-histidine tag-fused maltose-binding protein-monomeric streptavidin (6HISMBP-mSA-CGC). Anti-dig monoclonal antibody (mAb) and biotinylated-BSA are immobilized in the test and control line zones, respectively. Five plasma samples with known viral load (VL) were used to simulate the efficacy of early HIV-1 detection. RNA extracted from these samples was amplified by RT-PCR using the labeled primers, and DAB products were examined on agarose gel electrophoresis and LFA. RT-PCR from diluted clinical samples yielded visible DNA bands in agarose gel electrophoresis, consistent with positive LFA results. Conversely, negative samples only displayed the control line on LFA. This assay exhibited a limit of detection (LOD) of 82.29 RNA copies/mL, comparable to other nucleic acid amplification tests (NAATs). This novel technique provides a highly sensitive assay for early HIV-1 diagnosis, even with low VL, making it suitable for resource-limited settings.

Seven new mycoviruses identified from isolated ascomycetous macrofungi

Mycoviruses have been described in all major fungal taxonomic groups. There has been much focus on commercially cultivated basidiomycetous macrofungi, while attention to viruses from ascomycetous macrofungi is lacking. Therefore, in this study, we conducted viral screening against fungal mycelia that were regenerated from ascomycetous macrofungi using agarose gel electrophoresis (AGE) and fragmented and primer-ligated dsRNA sequencing (FLDS). Among the 57 isolates, four isolates were detected with virus-like bands through screening with AGE, and subsequent FLDS analyses determined the viral sequences. Other isolates without virus-like bands in AGE were pooled to check for viral sequences. Using FLDS analysis, a total of seven new mycoviruses were identified, including two double-stranded RNA (dsRNA) viruses belonging to Quadriviridae and Partitiviridae, five positive-sense single-stranded RNA (ssRNA) viruses (three belonging to Mitoviridae, one belonging to Endornaviridae and one belonging to Virgaviridae). All viruses characterized in this study are novel species, and all the hosts are firstly reported to be infected by mycoviruses. These findings expand our knowledge of the diversity of mycoviruses from macrofungi in natural environments.

A Pilot Study on Presence of Parkinson’s Disease Risk Gene PARK7 in Population of West Bengal, India: A Preliminary Observation

Introduction: The neurodegenerative Parkinson’s Disease (PD) is associated with different risk genes along with some environmental factors. Till date, there are few identified genes which play a pivotal role in the early onset of PD among the general population. Aim: To screen the presence of different risk genes of Parkinson’s disease along with associated environmental factors among randomly selected 62 urban participants. Materials and Methods: The present randomised control trial single blind pilot study was conducted in the Department of Biotechnology, Heritage Institute of Technology, Kolkata, West Bengal, India, from March 2021 to June 2021. The study participants were informed about the study and blood was collected from them. The genomic Deoxyribonucleic Acid (DNA) extraction was carried out following phenol chloroform extraction protocol and Polymerase Chain Reaction (PCR) was done with specific primers of the risk genes namely, Parkinson Disease 1 (PARK1) Alpha-synuclein (α-synuclein), Parkinson Disease 2 (PARK2) (Parkin), Parkinson Disease Protein 7 (PARK7) or DJ -1 and PINK 1. Protein Deglycase-1 (DJ-1) and PTEN Induced Kinase 1 (PINK1). The statistical analysis of the findings was done using Statistical package for the Social Sciences (SPSS) software version 21.0. Results: Out of 62 particpants, 47 were males and 15 were females and the age distribution of the participants was in between 21 years to >60 years. After obtaining specific band in agarose gel electrophoresis, 35 positive cases of PARK7 gene and 1 positive case of PARK2 gene were identified. Conclusion: There was a statistically significant association of different bacterial and viral infections with the PARK7 gene positive cases. PARK7 gene responsible for early onset of PD is significantly prevalent in the general population of West Bengal.

아가로스 겔 전기영동에서 간단하고 실용적인 DNA 염색 방법

Nucleic acid staining dyes are necessary for the visualization of DNA bands in agarose gel electrophoresis. The conventional staining method follows a post-staining protocol that follows gel electrophoresis; pre-cast staining is used as a convenient alternative. Pre-load staining is another method in which a staining dye is added to the 6X loading buffer, and not in the agarose gel. Pre-load staining is simple and time-saving; however, it could alter the mobility of DNA bands within the same gel. In this study, the mobility alteration owing to the use of commercial pre-mixed loading buffers, such as Midori Green Direct, GRGreen DNA loading buffer, and ENVISION DNA dye loading buffer, were evaluated. Lab-prepared 6X loading buffer containing 250X GelRed®dye did not alter the mobility under various experimental conditions. Similarly, 6X loading buffer containing Shinystar and EcodyeTMdid not affect mobility. In conclusion, pre-load staining method is a convenient and practical staining method for routine gel electrophoresis; lab-made 6X loading buffer containing 250X GelRed®could be used for pre-load staining.

Molecular detection and genotype diversity of hemoplasmas in non-hematophagous bats and associated ectoparasites sampled in peri-urban areas from Brazil

Hemoplasmas have already been detected in bats in the United States of America, Spain, Australia, Chile, Brazil, Peru, Belize, Nigeria, Costa Rica, Germany, Switzerland and New Caledonia. The recent detection of hemoplasmas closely related to Mycoplasma haematohominis, an agent causing disease in humans, emphasizes the need for additional studies on the diversity of hemoplasmas in bats. The present work aimed to investigate the occurrence and assess the phylogenetic positioning and genetic diversity of hemoplasmas in bats and associated ectoparasites sampled in central-western Brazil. Overall, 43% (58/135) sampled bats and 1.56% (1/64) bat flies (Megistopoda aranea) were positive for hemoplasmas, however, twenty-four and two hemoplasma sequences were obtained from PCR assays targeting 16S and 23S rRNA genes, respectively, since the majority of the obtained amplicons showed faint bands in agarose gel electrophoresis. The obtained 16S rRNA sequences showed to be broadly distributed along the phylogenetic tree, albeit positioned within the ‘Haemofelis group’ and clustering with other bat-associated hemoplasmas. Twelve 16S rRNA hemoplasma genotypes were found among the 24 obtained sequences. When compared to other bat-related hemoplasmas sequences retrieved from the Genbank, 52 genotypes were found. The two 23S rRNA sequences obtained were positioned as a sister clade to “Candidatus Mycoplasma haematohydrochaerus”, M. haemofelis and M. haemocanis. High genetic diversity was found among 16S rRNA hemoplasma sequences detected in non-hematophagous bats from central-western Brazil and previously detected in other regions of the world. Even though the genotype analysis showed that hemoplasmas from the same genus tend to group together, the results from the unipartite and bipartite analyses did not robustly support the hypothesis. Further studies addressing the specificity of hemoplasma genotypes according to bat species and genera should be performed.

Differential gene expression and immunohistochemical localization of the key melatonin biosynthesizing enzymes in the testis of zebrafish (Danio rerio)

ABSTRACT This study for the first time showed the presence of melatonin biosynthesizing genes (Tph1a, Aanat1, Aanat2 and Asmt) in the zebrafish (Danio rerio) testis. Melatonin is reported to be produced mainly in the pineal organ, but there is also evidence for the presence of melatonin in several extra-pineal sites. The presence of a single gene-specific band in agarose gel electrophoresis after end-point PCR and a single peak in qRT-PCR melting curve analysis indicates that the testis of zebrafish expresses the mRNA of enzymes for melatonin biosynthesizing machinery. Furthermore, we tried to demonstrate the presence of melatonin biosynthesizing enzymes at the translational level by immunohistochemistry, which confirms the localization of both Aanat and Asmt proteins in the testis of zebrafish. Moreover, melatonin ELISA reveals a higher level in the testis than the serum at noon, but a similar concentration is observed during the midnight. Moreover, testes display a rhythmic mRNA expression of Tph1, Aanat1, and Aanat2, while Asmt is arrhythmic. These results support an almost ubiquitous biosynthesis of melatonin in the peripheral organs of zebrafish, which can be related to a local role of this hormone as an autocrine or paracrine manner.

Identification and characterization of parvalbumin-like protein in Trichophyton violaceum

Parvalbumins play crucial physiological roles in neuromuscular systems of vertebrates, such as cell-cycle, development of neurons, contraction of muscles, and regulation of intracellular calcium. To perform these neuromuscular functions, parvalbumin may be in associated with other proteins including calbindin, carbonic anhydrase, and cytochrome oxidase. Humans may show an IgE-specific hypersensitivity to parvalbumins after consumption of some distinct fish species. While this protein is abundant in fish muscles, literature review of publications related to fish parvalbumins, do not point to the presence of parvalbumins in eukaryotic microbes. In this study, we propose that distantly related parvalbumins may be found in some non-fish species. Bioinformatics studies such as multiple sequence alignment (MSA), phylogenetic analysis as well as molecular-based experiments indicate that, at least two parvalbumins sequences (UniProt IDs: A0A178F775 and A0A178F7E4) with EF-hand domains and Ca2+-binding sites could be identified in Trichophyton violaceum, a pathogenic fungal species.It was determined that both genes consisted of a single exon and encoded for parvalbumin proteins possessing conserved amino acid motifs. Antigenicity prediction revealed antigenic sites located in both sides of the Ca2+-binding site of the first EF-hand domain. Our phylogenetic analysis revealed that one of parvalbumins (UniProt ID: 0A178F775) can be evolved to other parvalbumins in T. violaceum (UniProt ID: A0A178F7E4) and fish species through evolutionary phenomenon. To confirm our in-silico findings, we designed three primer pairs to detect one of the T. violaceum parvalbumins (UniProt ID: A0A178F7E4) by polymerase chain reaction (PCR); one primer pair showed a strong and specific band in agarose gel electrophoresis. To evaluate the specificity of the method, the primers were tested on extracted DNA from Trichophyton rubrum and T. mentagrophytes. The results demonstrated that the evaluated parvalbumin gene (UniProt ID: A0A178F7E4) was T. violaceum-specific and this pathogenic fungus can be differentiated from T. rubrum and T. mentagrophytes through identification of parvalbumin genes. Further studies are necessary to unravel the biochemical and physiological functions of parvalbumins in T. violaceum.

Identification and phylogeny of the small eukaryote population of raw and drinking waters

Culture-dependent and -independent methods were used to investigate the small eukaryote composition of raw and finished waters in the Norwegian cities of Oslo, Tromsø, Fredrikstad and Oppegård. Probes with general applicability to the 18S rRNA genes of the small eukaryote consortium were used for PCR-denaturing gradient gel electrophoresis (DGGE), and in the generation of clone libraries using the TOPO™ cloning and sequencing system. The chosen probes invariably gave a single band in agarose gel electrophoresis, indicating amplification of an area of similar size. DGGE and cloning analyses resolved the bands into components representing many unique amplicons. Diversity and composition in the collection were studied by DNA-sequencing, and visual examination of DGGE patterns. The cloning approach enabled the putative identification of a total of approximately 100 unique small eukaryotes. The major fraction of these represented ciliated and flagellated protozoal species. This was in keeping with the findings from protozoal cultivation. DNA from a number of multicellular eukaryotes was also detected. Amoebal and fungal DNA was rarely found. The latter may indicate a low incidence or a bias in the analysis technique. The population of small eukaryotes appears typical for pristine waters and no primary pathogens were detected by culture-independent techniques. However, the potentially pathogenic protozoa Acanthamoeba castellanii was grown on one occasion from Oslo’s drinking water. DGGE allowed the identification of fewer amplicons (by excision and sequencing of bands) than by the cloning-transformation approach. The DGGE analysis revealed clear similarities between the compositions of the raw and treated waters, indicating that cells or DNA in the raw water pass through the treatment trains. Protozoal culture and heterotrophic plate count analysis consistently revealed viable cells in both raw and treated waters in Oslo. This indicates that a fraction of the clone library represents eukaryotic species surviving the treatment trains. The analyses here presented represent the first published study of the general small eukaryotic fraction of the Capital’s drinking water, and those of three other Norwegian cities. We suggest that DGGE profiles may have a value in judging physical treatment efficacy (removal of cells), but that direct cloning and sequencing studies is more amenable for characterization of uncultured microbes.

Transcriptional Regulation of ATP2C1 Gene by Sp1 and YY1 and Reduced Function of its Promoter in Hailey–Hailey Disease Keratinocytes

Hailey-Hailey disease (HHD) is a blistering skin disease caused by malfunction of the Ca2+-dependent ATPase, ATP2C1. In this study, key regulatory regions necessary for the expression of the gene encoding human ATP2C1 were investigated. The transient reporter assay demonstrated that region +21/+57 was necessary for activation of the ATP2C1 promoter, and the electrophoretic mobility shift assay demonstrated that the region was recognized by the transcription factors, Sp1 and YY1. In accordance with this result, when Sp1 or YY1 was overexpressed in keratinocytes, an obvious increase in ATP2C1 promoter activity was observed, which was in contrast with the case where a mutant promoter lacking the binding sites for Sp1 and YY1 was used as the reporter. Ca2+-stimulation signal increased nuclear Sp1 proteins and ATP2C1 mRNA levels in normal keratinocytes. In contrast, both these increases were suppressed in keratinocytes from HHD patients. These results indicate that Sp1 and YY1 transactivate the human ATP2C1 promoter via cis-enhancing elements and that incomplete upregulation of ATP2C1 transcription contributes to the keratinocyte-specific pathogenesis of HHD. This is a report describing the regulation of the expression of ATP2C1.

A two-step method for the extraction of high-quality RNA from endoscopic biopsies.

The use of molecular techniques such as quantitative RT-PCR depends on the quality of cellular RNA. In particular, RNA extraction from endoscopic biopsies is difficult with respect to yield, and especially integrity. Endoscopic biopsies taken from the gastric antrum, corpus and duodenum were subjected to various RNA extraction protocols, and the RNA was used for quantitative RT-PCR. The subsequent use of two methods, (i) a phenol/chloroform extraction and (ii) a column-based extraction method, resulted in a yield of 4.5 microg total RNA per biopsy with reliable quality in 80% of samples. The quantitative RT-PCR analysis revealed that only RNA samples that clearly show both 18S- and 28S-RNA bands in agarose gel electrophoresis were suitable for quantitative RT-PCR as shown by expression of corpus-specific pepsinogen C-mRNA and the duodenum-specific multi-drug resistance protein-1 (mdr-1)-mRNA. In partially degraded RNA, pepsinogen C, mdr-1, or beta-actin mRNAs were still detectable, but the quantitative determination gave inconsistent data. The two-step method described here is a suitable option for extracting high-quality RNA from endoscopic biopsies when other standard protocols fail.

Quantitative Analysis of Plasmid Forms by Agarose and Capillary Gel Electrophoresis

Plasmids may appear in different forms: circular with different degrees of coiling, partially cleaved or linear, and multimeric as concatamers or catenates. Capillary gel electrophoresis (CGE) of plasmid samples allows the determination of plasmid form distribution. Monomeric and dimeric plasmid DNA forms were separated by both CGE and agarose gel electrophoresis (AGE). The pattern of isoform bands from AGE was compared to the corresponding peak pattern from CGE, and differences in the relative mobility of the plasmid forms between the two methods were found. The comparison of AGE and CGE allows the assignment of AGE bands to CGE peaks. Additionally, the different isoforms can now be quantified by CGE. Routine plasmid form analysis by CGE may be automated, allowing easy, fast, and highly reliable quantification. CGE also offers high resolution and the amount of DNA required is very low. Therefore this method is very useful for the analysis of therapeutics based on plasmid DNA during their production, isolation, and formulation.

Transcription from plasmid expression vectors is increased up to 14-fold when plasmids are transfected as concatemers.

A protocol for increasing transcription from plasmid expression vectors is presented. A vector containing chloramphenicol acetyltransferase (CAT) gene was digested leaving the transcription cassette intact. Heat inactivation of restriction enzymes followed by ligation of the digestion products yielded concatemers which migrated as a single band in agarose gel electrophoresis. Mouse fibroblasts transfected with the concatemers gave a CAT activity that was 14-fold greater than that of cells transfected with a similar mass (equimolar gene number) of the native plasmid. The effect was independent of promoter type, restriction enzyme, number of restriction sites and with a noted exception, cell line.

Immunoglobulin Conality Analysis:Resolution of Ambiguities in Immunofixation Electrophoresis Results by High-Resolution, Two-Dimensional Electrophoretic Analysis of Paraprotein Bands Eluted From Agarose Gels

This article describes evaluation of the clonality of origin of immunoglobulin (Ig) chains by strategies that include qualitative analysis of paraproteins with high-resolution, two-dimensional electrophoresis (2DE) and silver staining. This approach is helpful in evaluating specimens in which standard immunoelectrophoresis or immunofixation electrophoresis (IFE) techniques do not provide definitive Ig typing results. One method the authors developed involves a "band elution" procedure, in which proteins present in high-resolution agarose gel electrophoresis bands are cut from the agarose gel, eluted with a denaturing buffer, and subjected to 2DE. The microheterogeneity patterns of the Ig light chains, heavy chains, or both are evaluated for their relationship regarding mass, charge, and, by inference, number of genes of origin. When necessary, determination of charge relationships may be aided by urea-mediated carbamylation of lysine residues, which introduces single-charge shifts to the individual protein subunits. Overall, these adjunctive techniques are particularly useful in cases with multiple bands of identical immunologic types (eg, several IgG1 bands) on IFE before and after sulfhydryl reduction (with dithiothreitol or 2-mercaptoethanol). The authors present procedural details and examples of the 2DE band elution patterns of serum and urine samples from four patients with B-cell dyscrasias, including the first reported case of POEMS syndrome with biclonal gammopathy, to the best of their knowledge.

Transformation and expression of a staphylococcal plasmid in Escherichia coli.

A multiple antibiotic-resistant Staphylococcus aureus, was found to possess three plasmid bands in agarose gel electrophoresis. A plasmid of approximately 4.3 kb (pMC790/2) was found to code for ampicillin and tetracycline resistance and to have one EcoRI site when transformed into S. aureus RN 4220. pMC790/2 in unmodified form was transformed into a recA- E. coli at a frequency of 1.2 x 10(4) transformants/micrograms of plasmid DNA. Plasmid (pMC790/2) replicated, maintained itself stably and expressed far better in the E. coli host than in S. aureus.

Transformation and expression of a staphylococcal plasmid in Escherichia coli

Abstract A multiple antibiotic-resistant Staphylococcus aureus , was found to possess three plasmid bands in agarose gel electrophoresis. A plasmid of approximately 4.3 kb (pMC790/2) was found to code for ampicillin and tetracycline resistance and to have one Eco RI site when transformed into S. aureus RN 4220. pMC790/2 in unmodified form was transformed into a recA − E. coli at a frequency of 1.2×10 4 transformants/μg of plasmid DNA. Plasmid (pMC790/2) replicated, maintained itself stably and expressed far better in the E. coli host than in S. aureus .

High concentrations of eosinophil-derived neurotoxin in patients' urine mimic lysozyme far-cathodic bands in agarose gel electrophoresis

High concentrations of eosinophil-derived neurotoxin in patients' urine mimic lysozyme far-cathodic bands in agarose gel electrophoresis

Use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms

Rhinoviruses and enteroviruses are the major members of the picornavirus genus that cause human disease. We compared the polymerase chain reaction and viral culture for the identification of picornaviruses in nasal aspirates from children during episodes of respiratory symptoms and when asymptomatic and from asymptomatic adults. One hundred eight children, aged 9 to 11 years, completed a year-long study. Within 24 to 48 h of a report of respiratory symptoms, a nasal aspirate was taken in the home. Nasal aspirates were also taken from 65 of the children and from 33 normal adults when they had been free of respiratory symptoms for at least 2 weeks. Picornaviruses were isolated by culture for three passages in Ohio HeLa cells in rolling tubes at 33 degrees C and pH 7.0. For the polymerase chain reaction, duplicate 50-microliters samples were amplified with conserved primers from the 5' noncoding region. Picornaviruses generated approximately 380-bp bands in agarose gel electrophoresis; the specificity of these bands was confirmed by filter hybridization with a conserved internal probe. Picornaviruses were isolated by culture in 47 (46 rhinoviruses) of 292 symptomatic episodes (16%), whereas the polymerase chain reaction identified picornavirus genomic material in 146 episodes (50%), including all but one of the culture-positive episodes. As for asymptomatic samples, eight (12%) children and two (4%) adults were positive by the polymerase chain reaction, whereas only one child's specimen was positive by culture. This polymerase chain reaction assay represents a clear advance in the identification of picornavirus infection, with a detection rate threefold greater than the virus culture method.

Proteoglycan alterations following immobilization and remobilization in the articular cartilage of young canine knee (stifle) joint

The distribution of proteoglycans (PGs) at 11 sites on the knee (stifle joint) cartilage of young female beagle dogs was studied following cast immobilization for 11 weeks in 90 degrees flexion and after a subsequent remobilization for 15 weeks. Immobilization induced a reduction in PG uronic acid at all sites (mean of -38%), but the greatest depletion (-64%) occurred at the anterior and posterior extremes of the femoral condyles, i.e., at locations where the immobilized cartilage lost contact to the opposing cartilage. Following remobilization, the content of uronic acid remained lower than in the age-matched controls (-18% on average), particularly at the minimum contact sites most affected by immobilization (-33%). The chondroitin-6-sulfate to chondroitin-4-sulfate ratio was reduced by immobilization in most locations (average of -14%) and returned to control values after remobilization. There was no consistent change in the percentage of aggregating PGs observed in Sephacryl S-1000 gel filtration after immobilization or remobilization. However, following remobilization, the aggregating PGs showed an enhanced proportion of the slower mobility band in agarose gel electrophoresis, indicative of a larger monomer size. In the contralateral, load-bearing knee joint, both the uronic acid content and PG monomer type distribution were identical to those observed in the experimental joint, suggesting that the state reached after the remobilization period was due to factor(s) influencing both sides. The results suggest that contact forces between articulating surfaces are required to maintain normal PG content and that the control mechanism works locally at each cartilage site. Restriction of joint mobility and loading in young animals is concluded to cause persistent changes in cartilage matrix. Furthermore, the use of the contralateral joint as the sole control in this kind of studies, although experimentally convenient, seems not to be appropriate.

A biological role of the carbohydrate moieties of laminin.

The ways in which the carbohydrate moieties of laminin affect its cellular interactions have been examined by two different experimental approaches. In one approach, we used lectins in order to block specific carbohydrates on laminin which previously had been dried onto a plastic surface. We found that wheat germ agglutinin and Griffonia simplicifolia agglutinin I blocked the binding of the neuron-like rat pheochromocytoma cell line PC12. However, when concanavalin A was used cell binding was unaffected but neurite outgrowth was prevented, compared to controls, over a 24-h period. In the second approach we used unglycosylated laminin as a substratum on the plastic surface. We have developed a method for the purification of unglycosylated laminin from tunicamycin treated cultures of a mouse embryonal carcinoma derived cell line, M1536 B3, and have partially characterized the purified material. A mixture of unglycosylated and glycosylated laminin was selectively purified from the M1536 B3 cell lysate by an anti-EHS laminin monoclonal antibody immunoaffinity column. The unglycosylated laminin was separated from glycosylated laminin using G. simplicifolia lectin affinity chromatography. The lectins, wheat germ agglutinin, G. simplicifolia agglutinin I, and concanavalin A, did not bind to any of the subunits of unglycosylated laminin in Western blots. The unglycosylated laminin migrated as a single band in agarose-gel electrophoresis under nonreducing conditions indicating that it is a fully assembled and disulfide bonded molecule. Circular dichroism studies showed no differences between glycosylated and unglycosylated laminin, indicating similar molecular conformations. Western blots using antibodies specific for the A, B1, and B2 chains of laminin showed that unglycosylated laminin contained each of these subunits. We then performed cell binding and spreading or neurite outgrowth assays using unglycosylated laminin. A mouse melanoma cell line, B16 F1, bound to this laminin in the same numbers as to the control glycosylated laminin, but cell spreading was minimal. When this unglycosylated laminin was used as a substrate for PC12 cells neurite outgrowth was impaired; no effect was noted on the number of cells bound, compared to glycosylated laminin. We conclude from these results that once cells become bound to laminin the carbohydrate residues of that glycoprotein must be available to enable the cells to spread or to extend neurite processes.