scholarly journals Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259846
Author(s):  
Yasuhiro Fujiwara ◽  
Yuji Tanno ◽  
Hiroki Sugishita ◽  
Yusuke Kishi ◽  
Yoshinori Makino ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Concanavalin A ◽  
Magnetic Particles ◽  
Magnetic Beads ◽  
Protein A ◽  
Con A ◽  
Established Method ◽  
Genomic Location ◽  
Tn5 Transposase ◽  
Selection Of

Epigenome research has employed various methods to identify the genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapter sequences nearby the target protein. Throughout most of the CUT&Tag procedure, cells are held on concanavalin A (con A)-conjugated magnetic beads. Proper holding of cells would be decisive for the accessibility of Tn5 to the chromatin, and efficacy of the procedure of washing cells. However, BioMag®Plus ConA magnetic beads, used in the original CUT&Tag protocol, often exhibit poor suspendability and severe aggregation. Here, we compared the BioMag beads and Dynabeads® magnetic particles of which conjugation of con A was done by our hands, and examined the performance of these magnetic beads in CUT&Tag. Among tested, one of the Dynabeads, MyOne-T1, kept excessive suspendability in a buffer even after overnight incubation. Furthermore, the MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3. In conclusion, the arrangement and the selection of MyOne-T1 refine the suspendability of beads, which improves the association of chromatin with Tn5, which enhances the sensitivity in CUT&Tag assay.

scholarly journals Preparation of "stress-free" concanavalin A-conjugated Dynabeads magnetic beads for CUT&Tag

2021 ◽  
Author(s):  
Yasuhiro Fujiwara ◽  
Yuji Tanno ◽  
Hiroki Sugishita ◽  
Yusuke Kishi ◽  
Yoshinori Makino ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Concanavalin A ◽  
Magnetic Particles ◽  
Magnetic Beads ◽  
Protein A ◽  
Con A ◽  
Established Method ◽  
Genomic Location ◽  
Tn5 Transposase ◽  
Selection Of

Epigenome research has employed various methods to identify genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapter sequences nearby the target protein. Throughout most of the CUT&Tag procedure, cells are held on concanavalin A (con A)-conjugated magnetic beads. Proper holding of cells would be decisive for the accessibility of Tn5 to the chromatin, and efficacy of the procedure of washing cells. However, BioMagPlus ConA magnetic beads, used in the original CUT&Tag protocol, often exhibit poor suspendability and severe aggregation. Here, we compared the BioMag beads and Dynabeads magnetic particles of which conjugation of con A was done by our hands, and examined the performance of these magnetic beads in CUT&Tag. Among tested, one of the Dynabeads, MyOne-T1, kept excessive suspendability in a buffer even after overnight incubation. Furthermore, the MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3. In conclusion, the arrangement and the selection of MyOne-T1 refine the suspendability of beads, which improves the association of chromatin with Tn5, which enhances the sensitivity in CUT&Tag assay.

scholarly journals Antibody-guided Chromatin Tagmentation for Two or More Factors (ACT2-seq)

2021 ◽  
Author(s):  
Benjamin Carter ◽  
Wai Lim Ku ◽  
Keji Zhao
Keyword(s):  
Single Cell ◽  
Magnetic Beads ◽  
Protein A ◽  
Epigenetic Marks ◽  
Tn5 Transposase

Abstract This protocol details the reagents and steps required to perform antibody-guided chromatin tagmentation for two or more factors (ACT2-seq, ACT2). Like its predecessor ACT-seq, ACT2 uses a fusion of protein A and Tn5 transposase to bind and profile epigenetic marks across the genome. ACT2 builds on the capabilities of ACT-seq by directly and concurrently profiling co-occupancy of epigenetic marks, which previously required laborious, expensive, and technically challenging approaches involving fluorescence, magnetic beads, or single-cell methods. ACT2 requires only standard pipetting and centrifugation techniques and can be completed in less than a single day of bench work.

scholarly journals Interactions between human eosinophils and schistosomula of schistosoma mansoni. II. The mechanism of irreversible eosinophil adherence

1979 ◽  
Vol 150 (6) ◽  
pp. 1456-1471 ◽  
Author(s):  
AE Butterworth ◽  
MA Vadas ◽  
DL Wassom ◽  
A Dessein ◽  
M Hogan ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Concanavalin A ◽  
Protein A ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Con A ◽  
Normal Human ◽  
Culture Supernate ◽  
Eosinophil Major Basic Protein

Previous work (1)(1) has shown that normal human eosinophils show a preferential capacity, in comparison with neutrophils, to bind to antibody- coated schistosomula of Schistosoma mansoni. This effect is attributable to a temperature-dependent function of the eosinophil which renders its binding stable and irreversible by aggregated gamma globulin or Staphylococcus aureus protein A. In contrast, the binding of neutrophils is readily reversible by these agents. It has now been shown that the differences observed between eosinophils and neutrophils is a property of their interaction with living schistosomula. When dead or artificially damaged schistosomula were tested, neutrophils showed a markedly enhanced capacity to adhere, in both the presence and absence of anti-chistosomular serum. Subsequent experiments were designed to test the hypothesis that the strong, stable binding of eosinophils was attributable to degranulation, with release of granule contents which would then serve as ligands to bind the cell to the organism. First, an enhanced adherence both of eosinophils and of neutrophils could be demonstrated in the presence of eosinophil major basic protein (MBP) or of protamine, a high molecular weight cation. Second, the binding of eosinophils induced by concanavalin A (Con A) was found to differ markedly from that induced by antischistosomular serum. Con A-mediated binding of eosinophils was fully reversible by alpha-methyl-mannoside, was not associated with damage to the organism, and did not lead to degranulation of the cell, as estimated by measuring the release of MBP into the culture supernate. However, induction of degranulation of concanavalin A-bound eosinophils, but not of neutrophils, with the calcium ionophore A23187 converted the reaction into one which was no longer reversible by alpha- methylmannoside and in which damage to the organism now did occur. These findings support the hypothesis that the stable binding of eosinophils is associated with degranulation, a process which may contribute to the preferential capacity of this cell to mediate antibody-dependent damage to schistosomula.

Application of Concanavalin A–Linked Magnetic Beads for the Detection of Hepatitis A Virus

2018 ◽  
Vol 81 (12) ◽  
pp. 1997-2002
Author(s):  
SANG-MU KO ◽  
SE-YOUNG CHO ◽  
MYUNG-JOO OH ◽  
JOSEPH KWON ◽  
BIPIN VAIDYA ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Binding Affinity ◽  
Concanavalin A ◽  
Hepatitis A ◽  
Magnetic Beads ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Carbohydrate Binding ◽  
Con A ◽  
Reverse Transcriptase Pcr

ABSTRACT Prompt and inexpensive detection of hepatitis A virus (HAV) is essential to control acute hepatitis outbreaks associated with the consumption of contaminated raw or minimally processed food. In this study, various carbohydrate-binding lectins, including concanavalin A (Con A), wheat germ agglutinin, and soybean agglutinin, were compared for their binding affinity to HAV. Con A, which showed significantly higher binding affinity than other lectins, was used to develop an alternative and affordable method to conventional antibody-linked immunomagnetic separation prior to detection of HAV using reverse transcriptase PCR. This method, Con A–linked immunomagnetic separation combined with reverse transcriptase PCR, can detect HAV at a dilution concentration of 10−4 of the virus stock (titer: 104 median tissue culture infective dose per mL), indicating that Con A could be a promising candidate for concentrating HAV.

GAGA protein: a multi-faceted transcription factorThis paper is one of a selection of papers published in this Special Issue, entitled 27th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

2006 ◽  
Vol 84 (4) ◽  
pp. 559-558 ◽  
Author(s):  
Nicholas L. Adkins ◽  
Thomas A. Hagerman ◽  
Philippe Georgel
Keyword(s):  
Review Process ◽  
Peer Review Process ◽  
Protein A ◽  
Gaga Factor ◽  
Nucleosome Remodeling ◽  
Regulate Gene Expression ◽  
Genomic Location ◽  
Multiple Levels ◽  
Repressor Activity ◽  
Selection Of

The transition from transcription activation to repression is regulated at multiple levels by the DNA sequence and DNA modification to its compaction through chromatin packaging. The GAGA factor (GAF) is one of a few transcription factors that can regulate gene expression at multiple levels. It displays both activator/antirepressor and repressor activity, depending on its target genomic location. The GAF-mediated modulation of expression appears to be intimately linked with modifications of the chromatin structure. The GAF can associate with highly compacted heterochromatin, contributing to gene repression, or participate in nucleosome remodeling to activate specific genes. In this review, we are attempting to elucidate the contribution(s) of the various domains of the GAF to the recruitment of its functional partners, leading to seemingly opposite functions. We surveyed the current scientific literature for evidence of GAF involvement in regulatory events associated with changes of chromatin composition or conformation.

Concanavalin A-induced Aggregation of Human Blood Platelets

1975 ◽  
Vol 33 (02) ◽  
pp. 354-360 ◽  
Author(s):  
Heinrich Patscheke ◽  
Reinhard Brossmer
Keyword(s):  
Concanavalin A ◽  
Binding Capacity ◽  
Specific Binding ◽  
Blood Platelets ◽  
Residual Effect ◽  
Independent Effect ◽  
Con A ◽  
Experimental Conditions ◽  
Main Effect ◽  
Induced Aggregation

SummaryConcanavalin A (CON A) causes platelets to aggregate. A Ca++-independent effect of CON A could be separated from a main effect which depends on Ca++. The main effect probably is a consequence of the CON A-induced platelet release reaction and therefore is platelet-specific. The weak residual effect observed in the presence of Na2EDTA may be due to a similar mechanism as has been demonstrated for CON A-induced aggregations of several other normal and malignant transformed animal cells.Na2EDTA did not inhibit the carbohydrate-specific binding capacity of CON A. Therefore, Na2EDTA appears not to demineralize the CON A molecules under these experimental conditions.α-methyl-D-glucoside inhibits the Ca++-independent as well as the Ca++-dependent effect of CON A.Pretreatment by neuraminidase stimulated the platelet aggregation induced by CON A. It is possible that removal of terminal sialic acid residues makes additional receptors accessible for the binding of CON A.

scholarly journals MicroRNA Mediating Networks in Granulosa Cells Associated with Ovarian Follicular Development

2017 ◽  
Vol 2017 ◽  
pp. 1-18 ◽  
Author(s):  
Baoyun Zhang ◽  
Long Chen ◽  
Guangde Feng ◽  
Wei Xiang ◽  
Ke Zhang ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Granulosa Cells ◽  
Messenger Rna ◽  
Expression Patterns ◽  
Follicular Development ◽  
Functional Networks ◽  
Signal Pathways ◽  
Differentially Expressed Mirnas ◽  
Selection Of

Ovaries, which provide a place for follicular development and oocyte maturation, are important organs in female mammals. Follicular development is complicated physiological progress mediated by various regulatory factors including microRNAs (miRNAs). To demonstrate the role of miRNAs in follicular development, this study analyzed the expression patterns of miRNAs in granulosa cells through investigating three previous datasets generated by Illumina miRNA deep sequencing. Furthermore, via bioinformatic analyses, we dissected the associated functional networks of the observed significant miRNAs, in terms of interacting with signal pathways and transcription factors. During the growth and selection of dominant follicles, 15 dysregulated miRNAs and 139 associated pathways were screened out. In comparison of different styles of follicles, 7 commonly abundant miRNAs and 195 pathways, as well as 10 differentially expressed miRNAs and 117 pathways in dominant follicles in comparison with subordinate follicles, were collected. Furthermore, SMAD2 was identified as a hub factor in regulating follicular development. The regulation of miR-26a/b onsmad2messenger RNA has been further testified by real time PCR. In conclusion, we established functional networks which play critical roles in follicular development including pivotal miRNAs, pathways, and transcription factors, which contributed to the further investigation about miRNAs associated with mammalian follicular development.

Magnetophoresis ‘meets’ viscoelasticity: deterministic separation of magnetic particles in a modular microfluidic device

Lab on a Chip ◽  
2015 ◽  
Vol 15 (8) ◽  
pp. 1912-1922 ◽  
Author(s):  
Francesco Del Giudice ◽  
Hojjat Madadi ◽  
Massimiliano M. Villone ◽  
Gaetano D'Avino ◽  
Angela M. Cusano ◽  
...  
Keyword(s):  
Microfluidic Device ◽  
Magnetic Particles ◽  
Magnetic Beads ◽  
Microfluidic Channel

Deflection of magnetic beads in a microfluidic channel can be improved through viscoelastic focusing.

scholarly journals Visualization of intracellular concanavalin A binding sites in retinal photoreceptors.

1978 ◽  
Vol 26 (10) ◽  
pp. 822-828 ◽  
Author(s):  
I Nir
Keyword(s):  
Concanavalin A ◽  
Binding Sites ◽  
Photoreceptor Cells ◽  
Con A ◽  
Thin Sections ◽  
Glycol Methacrylate ◽  
Outer Segments ◽  
Retinal Photoreceptors ◽  
Carbohydrate Components ◽  
The Relationship

Localization of carbohydrate components in retinal photoreceptor cells and membranes was studied. Frog and rat retinas were fixed with glutaraldehyde and embedded in glycol methacrylate or in a mixture of glycol methacrylate, glutaraldehyde and urea. Thin sections were incubated with ferritin-labeled concanavalin A (F-Con A) and stained with osmium vapors. Intensive binding was observed in both rod and cone outer segments. In the rod inner segment, differential binding of F-Con A was demonstrated. While numerous ferritin granules were observed in the myoid zone, only a few were seen in the ellipsoid zone, except for a local accumulation along the plasma membrane. In the rod outer segment, Con A binding sites were closely associated with the disk membranes. Ferritin granules were observed on both sides of the membranes. The relationship between the localization of Con A binding sites and the orientation of visual pigment molecules within the rod outer segments disk membranes was discussed.

EGF receptor signaling induces pointed P1 transcription and inactivates Yan protein in the Drosophila embryonic ventral ectoderm

Development ◽  
1996 ◽  
Vol 122 (11) ◽  
pp. 3355-3362 ◽  
Author(s):  
L. Gabay ◽  
H. Scholz ◽  
M. Golembo ◽  
A. Klaes ◽  
B.Z. Shilo ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Egf Receptor ◽  
Protein A ◽  
Drosophila Embryo ◽  
Cell Fates ◽  
Secondary Target ◽  
Graded Activity ◽  
Definition Of

The induction of different cell fates along the dorsoventral axis of the Drosophila embryo requires a graded activity of the EGF receptor tyrosine kinase (DER). Here we have identified primary and secondary target genes of DER, which mediate the determination of discrete ventral cell fates. High levels of DER activation in the ventralmost cells trigger expression of the transcription factors encoded by ventral nervous system defective (vnd) and pointed P1 (pntPl). Concomitant with the induction of pntP1, high levels of DER activity lead to inactivation of the Yan protein, a transcriptional repressor of Pointed-target genes. These two antagonizing transcription factors subsequently control the expression of secondary target genes such as otd, argos and tartan. The simultaneous effects of the DER pathway on pntP1 induction and Yan inactivation may contribute to the definition of the border of the ventralmost cell fates.

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