Electrophoretic transfer of RNA to nylon membrane (Semi-dry blotting) v1

protocols.io ◽  
2017 ◽  
Author(s):  
Anna Behle
Keyword(s):  
Nylon Membrane ◽  
Electrophoretic Transfer

scholarly journals Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides

2016 ◽  
Vol 2016 ◽  
pp. 1-4
Author(s):  
Chun Wu
Keyword(s):  
Dna Polymerase ◽  
Click Reaction ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Sequencing Analysis ◽  
Abasic Site ◽  
Nylon Membrane ◽  
Abasic Sites ◽  
Polymerase Chain ◽  
Dna Sequencing Analysis ◽  
Endonuclease Digestion

Biotinylation of deoxyguanosine at an abasic site in double-stranded oligodeoxynucleotides was studied. The biotinylation of deoxyguanosine is achieved by copper-catalyzed click reaction after the conjugation of the oligodeoxynucleotide with 2-oxohex-5-ynal. The biotinylation enables visualization of the biotinylated oligodeoxynucleotides by chemiluminescence on a nylon membrane. In order to investigate the biotinylated site, the biotinylated oligodeoxynucleotides were amplified by the DNA polymerase chain reaction. Replacement of guanine opposing the abasic site with adenine generated by the activity of the terminal deoxynucleotidyl transferase of DNA polymerase was detected by DNA sequencing analysis and restriction endonuclease digestion. This study suggests that 2-oxohex-5-ynal may be useful for the detection of the unpaired deoxyguanosine endogenously generated at abasic sites in genomic DNA.

scholarly journals Affinity purification and subunit structures of β-N-acetylhexosaminidases A and B from boar epididymis

1984 ◽  
Vol 219 (3) ◽  
pp. 1009-1015 ◽  
Author(s):  
H C Parkes ◽  
J L Stirling ◽  
P Calvo
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B ◽  
Electrophoretic Transfer ◽  
Peptide Maps

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.

scholarly journals Widespread Occurrence of Cotton leaf curl virus on Radish in Pakistan

Plant Disease ◽  
2000 ◽  
Vol 84 (7) ◽  
pp. 809-809 ◽  
Author(s):  
S. Mansoor ◽  
S. Mukhtar ◽  
M. Hussain ◽  
I. Amin ◽  
Y. Zafar ◽  
...  
Keyword(s):  
Host Range ◽  
Full Length ◽  
Cotton Leaf ◽  
Degenerate Primers ◽  
Nylon Membrane ◽  
Punjab Province ◽  
Cotton Leaf Curl Virus ◽  
Kitchen Gardens ◽  
Leaf Curl Virus ◽  
Leaf Curl

The current epidemic of cotton leaf curl disease (CLCuD) in Pakistan started in 1988 with the natural host range limited to a few plant species in the family Malvaceae. However, we have observed expansion in the host range of the virus, and several non-Malvaceous plants were found to be infected with the virus. Characteristic symptoms of CLCuD such as leaf curl and enations have been observed on radish plants, primarily in kitchen gardens. However, in 1999, levels of infection of 10 to 90% were observed both in commercial fields and kitchen gardens in the Punjab province of Pakistan. Both symptomatic and nonsymptomatic samples were collected from five different locations. Total DNA was isolated, dot-blotted on nylon membrane, and a full-length clone corresponding to DNA A of cotton leaf curl virus was labeled with 32P dCTP and used as a probe for the detection of a begomovirus. Strong signals were observed in symptomatic plants while no signals were observed in nonsymptomatic plants. Infection with a begomovirus was further confirmed by polymerase chain reaction (PCR) using degenerate primers for DNA A (1). Primers specific for the two distinct begomoviruses associated with CLCuD were also used in PCR reactions (2), and products of the expected size were obtained from all symptomatic samples, confirming infection with begomoviruses similar to those associated with CLCuD. A full-length probe of a nanovirus-like molecule associated with cotton leaf disease (3), called DNA 1 was labeled with 32P dCTP and detected the virus only in symptomatic plants. Similarly, primers specific for DNA 1 (3) amplified a product of expected size when used in PCR. On the basis of symptomatology and the detection of specific viral components associated with the disease, we confirmed that radish plants are infected with Cotton leaf curl virus (CLCuV). Since radish is a short duration crop, infection of CLCuV in radish may not serve as a direct source of infection for the next cotton crop. However, it is a potential threat to tomato crops which overlap with radish in the Punjab province. The detection of CLCuD in radish is another example of the mobilization of begomoviruses to previously unknown hosts. References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) S. Mansoor et al. Pak. J. Bot. 31:115, 1999. (3) Mansoor et al. Virology 259:190, 1999.

scholarly journals Regulation of complement functional efficiency by histidine-rich glycoprotein

Blood ◽  
1992 ◽  
Vol 79 (11) ◽  
pp. 2973-2980 ◽  
Author(s):  
NS Chang ◽  
RW Leu ◽  
JA Rummage ◽  
JK Anderson ◽  
JE Mole
Keyword(s):  
Divalent Cations ◽  
Alternative Pathway ◽  
Normal Serum ◽  
Peptide Sequence ◽  
Poly I:C ◽  
Complement Alternative Pathway ◽  
Nylon Membrane ◽  
Complement Components ◽  
S Protein ◽  
Functional Efficiency

The modulation of complement functional efficiency by serum histidine- rich glycoprotein (HRG) was investigated. Addition of exogenous HRG to prewarmed diluted serum, followed immediately by sensitized sheep erythrocytes (EA), resulted in enhanced hemolysis. However, when HRG was incubated with diluted serum for 10 minutes at 37 degrees C, inhibition of hemolysis occurred. The biphasic modulation of complement function was also obtained with the complement alternative pathway when HRG was added to diluted serum for hemolysis of rabbit erythrocytes. Partial reduction of complement functional activity was shown when serum was absorbed by an HRG-Sepharose 6MB column. Western blot analysis showed that complement C8, C9, factor D, and S-protein in diluted serum were bound by nylon membrane-immobilized HRG. However, by immunoprecipitation of relatively undiluted serum with anti-HRG IgG beads, HRG was found to coprecipitate with S-protein and plasminogen, which suggested that HRG may complex with these proteins in serum. In functional tests, HRG inhibited C8 hemolytic activity, probably by preventing C8 binding to EAC1–7 cells. HRG also enhanced polymerization of purified C9 as well as the generation of a 45-Kd C9 fragment. Such an effect was even more pronounced in the presence of divalent cations with the reaction mixtures of C9 and HRG. Partial dimerization of C9 was shown when exogenous HRG was added to normal serum. In contrast, polymerization of serum C9 was inhibited by exogenous HRG during poly I:C activation of serum or incubation under low ionic strength conditions. HRG was further shown to inhibit factor D-mediated cleavage of factor B when bound by cobra venom factor. The molecular basis by which HRG regulates serum complement function is not clear. Hypothetically, the tandem repetitions of a consensus histidine-rich penta-peptide sequence in HRG may provide a highly charged area that interacts with complement components.

Interaction of N atoms through a nylon membrane in nitrogen flowing post discharges

2006 ◽  
Vol 39 (17) ◽  
pp. 3826-3830 ◽  
Author(s):  
S Villeger ◽  
M Sixou ◽  
J Durand ◽  
A Ricard
Keyword(s):  
Nylon Membrane

Digoxigenin-Labeled Probe-Based Colony Blotting Assay for Rapid Quantification of Salmonella Serovars in Seafood and Water

2012 ◽  
Vol 95 (6) ◽  
pp. 1652-1655 ◽  
Author(s):  
Rakesh Kumar ◽  
K V Lalitha
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Gene Probe ◽  
Pcr Assay ◽  
Nylon Membrane ◽  
Rapid Enumeration ◽  
Salmonella Serovars ◽  
Labeled Probe

Abstract A non-radio-labeled probe-based detection method was developed for rapid enumeration of Salmonella in seafood and water samples. A Salmonella-specific invA gene probe was developed using a digoxigenin-based non-radio labeling assay, which was evaluated with naturally contaminated seafood and water samples. The probe-based technique was further compared with the quantitative PCR assay. The method was specific for detection of different Salmonella serovars without any nonspecific hybridization with other Salmonella-related Enterobacteriaceae. The optimum labeling efficiency was determined for the labeled probe, and 10 pg/μL probe concentration was observed to be most efficient for detection of Salmonella colonies on nylon membrane. Quantification of Salmonella in naturally contaminated seafood and water samples (n = 21) was in the range 10–102 CFU/mL. The assay successfully quantified Salmonella in spiked seafood and water samples in the presence of background flora, and the entire assay was completed within 48 h. The probe-based assay was further evaluated with real-time PCR, and results showed that the assay was comparable to real-time PCR assay. Thus, this probe-based assay can be a rapid, useful, and alternative technique for quantitative detection of Salmonella in food, feed, and water samples.

Variables that Affect Immobilization of Mucor Miehei Lipase on Nylon Membrane

2004 ◽  
Vol 20 (4) ◽  
pp. 371-375 ◽  
Author(s):  
Laura Maria Bruno ◽  
Gustavo Adolfo Saavedra Pinto ◽  
Heizir Ferreira de Castro ◽  
José Luiz de Lima-Filho ◽  
Eduardo Henrique de Magalhães Melo
Keyword(s):  
Nylon Membrane ◽  
Mucor Miehei ◽  
Mucor Miehei Lipase ◽  
Miehei Lipase

Alginate dialdehyde meets nylon membrane: a versatile platform for facile and green fabrication of membrane adsorbers

2018 ◽  
Vol 6 (11) ◽  
pp. 1640-1649 ◽  
Author(s):  
M. Kamran Khan ◽  
Jianquan Luo ◽  
Zhaoshuai Wang ◽  
Rashid Khan ◽  
Xiangrong Chen ◽  
...  
Keyword(s):  
Intermediate Layer ◽  
Nylon Membrane ◽  
Biocompatible Polymer ◽  
Membrane Adsorbers

Alginate dialdehyde, a biocompatible polymer, is used as an intermediate layer on a nylon membrane to readily fabricate different membrane adsorbers.

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