In-Yeast Meeting of Coronavirus Infectious cDNA Clones Utilizing a Artificial Genomics Pipeline
The Escherichia coli and vaccinia virus-based reverse genetics methods have been broadly utilized for the manipulation and engineering of coronavirus genomes. These methods, nonetheless, current a number of limitations and are generally tough to determine in a well timed method for (re-)rising viruses. On this chapter, we current a brand new common reverse genetics platform for the meeting and engineering of infectious full-length cDNAs utilizing yeast-based transformation-associated recombination cloning.
This novel meeting technique not solely ends in secure coronavirus infectious full-length cDNAs cloned within the yeast Saccharomyces cerevisiae but additionally fosters and accelerates the manipulation of their genomes.
Such a platform is broadly relevant for the scientific neighborhood, because it requires no particular gear and could be carried out in an ordinary laboratory setting. The protocol described could be simply tailored to just about all recognized or rising coronaviruses, reminiscent of Center East respiratory syndrome coronavirus (MERS-CoV).
RNA-cDNA hybrids mediate transposition via different mechanisms
Retrotransposons can represent half of eukaryotic genomes. Retrotransposon dysregulation destabilizes genomes and has been linked to various human diseases. Emerging regulators of retromobility include RNA-DNA hybrid-containing structures known as R-loops. Accumulation of these structures at the transposons of yeast 1 (Ty1) elements has been shown to increase Ty1 retromobility through an unknown mechanism.
Here, via a targeted genetic screen, we identified the rnh1Δ rad27Δ yeast mutant, which lacked both the Ty1 inhibitor Rad27 and the RNA-DNA hybrid suppressor Rnh1. The mutant exhibited elevated levels of Ty1 cDNA-associated RNA-DNA hybrids that promoted Ty1 mobility. Moreover, in this rnh1Δ rad27Δ mutant, but not in the double RNase H mutant rnh1Δ rnh201Δ, RNA-DNA hybrids preferentially existed as duplex nucleic acid structures and increased Ty1 mobility in a Rad52-dependent manner.
The data indicate that in cells lacking RNA-DNA hybrid and Ty1 repressors, elevated levels of RNA-cDNA hybrids, which are associated with duplex nucleic acid structures, boost Ty1 mobility via a Rad52-dependent mechanism.
In contrast, in cells lacking RNA-DNA hybrid repressors alone, elevated levels of RNA-cDNA hybrids, which are associated with triplex nucleic acid structures, boost Ty1 mobility via a Rad52-independent process. We propose that duplex and triplex RNA-DNA hybrids promote transposon mobility via Rad52-dependent or -independent mechanisms.
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Identification of salinity responsive genes in lavender through cDNA-AFLP
Currently, a global demand exists forlavender as a significant medicinal plant and source of essential oils. Freshwater and arable lands are two major factors that inhibit extensive farming of medicinal plants in Iran. Saline water from seas and salty soil may be new resources for agricultural use, especially for medicinal plants.
We sought to extend our knowledge of the Lavandula angustifolia genome and molecular basis of its salinity tolerance by using cDNA amplified fragment length polymorphism (cDNA-AFLP) to investigate the changes in plant transcriptomes in response to NaCl.
All identified transcript derived fragments (TDF) were assigned as novel L. angustifolia genes related to signal transduction, regulation of gene expression, alternative splicing, autophagy, and secondary metabolite biosynthesis. qRT-PCR analysis of the TDFs in response to different concentrations of NaCl revealed various levels of mRNA of the identified genes in this plant. Our findings provided primary insights into the molecular response of L. angustifolia to salinity.
Description: Human Histone 3 (HIST1H3E), GenBank Accession No. NM_003532, a.a. 2-136(end) with N-terminal His-tag MW = 15.4 kDa, expressed in an E. coli expression system.
Description: Human Histone 3 (HIST1H3E), GenBank Accession No. NM_003532, a.a. 2-136(end) with N-terminal His-tag MW = 15.4 kDa, expressed in an E. coli expression system.
Description: Human Histone H1, also known as H1F0, (GenBank Accession No. NM_005318), a.a. 2-194(end) with N-terminal His-tag, MW = 21.7 kDa, expressed in an E. coli expression system.
Description: Human Histone H1, also known as H1F0, (GenBank Accession No. NM_005318), a.a. 2-194(end) with N-terminal His-tag, MW = 21.7 kDa, expressed in an E. coli expression system.
Recombinant (Sf9) HIV-1 p24 Core mosaic protein (p24 full length, plus CT of p17 and NT of p15)
Description: Protein tyrosine phosphatase PTP1B, also known as PTPN1 (GenBank accession number M31724), full length, (a.a. 1-436), with N terminal GST-tag, MW=76 kDa, expressed in an E. coli expression system.
Description: Human Histone 2A, GenBank Accession No. NM_033445, a.a. 2-130(end) with N-terminal His-tag, MW = 14.8 kDa, expressed in an E. coli expression system.
Description: Human Histone 2B, GenBank Accession No. NM_003528, a.a. 2-126(end) with N-terminal His-tag, MW = 14.6 kDa, expressed in an E. coli expression system. _x000D_
Description: Human Histone 2A, GenBank Accession No. NM_033445, a.a. 2-130(end) with N-terminal His-tag, MW = 14.8 kDa, expressed in an E. coli expression system.
Description: Human Histone 2B, GenBank Accession No. NM_003528, a.a. 2-126(end) with N-terminal His-tag, MW = 14.6 kDa, expressed in an E. coli expression system. _x000D_
Description: Human JARID1B, also_x000D_ known as KDM5B and PLU-1 (GenBank_x000D_ Accession No. NM_006618), a.a. 2-_x000D_1544(end) with N-terminal FLAG-Avi-tag,_x000D_ MW=179 kDa, expressed in Sf9 cells via_x000D_ a baculovirus expression system.
Description: Human recombinant histone octamer consisting of 2 molecules each of histones H2A (a.a. 2-130(end)), H2B (a.a. 2-126(end)), H3 (a.a. 2-137(end)), and H4 (a.a. 2-103(end)), expressed in an E. coli expression system. GenBank Accession Nos. are NM_033445, NM_003528, NM_003532, and NM_003548, respectively. Each histone protein has an N-terminal His-tag. Global MW = 113.8 kDa._x000D_
Description: Human recombinant histone H3 (a.a. 1-136(end)) with K27C, C97A, and C111A mutations, and an N-terminal His-tag, expressed in an E. coli expression system. GenBank Accession No. NM_003532. The protein has been alkylated to produce the lysine analog, aminoethylcysteine. MW = 16 kDa.
Description: Human recombinant histone H3 (a.a. 1-136(end)) with K27C, C97A, and C111A mutations, and an N-terminal His-tag, expressed in an E. coli expression system. GenBank Accession No. NM_003532. The protein has been alkylated to produce the lysine analog, methyl aminoethylcysteine. MW = 16 kDa.
Description: Human recombinant histone H3 (a.a. 1-136(end)) with K27C, C97A, and C111A mutations, and an N-terminal His-tag, expressed in an E. coli expression system. GenBank Accession No. NM_003532. The protein has been alkylated to produce the lysine analog, dimethyl aminoethylcysteine. MW = 16 kDa.
Description: Human recombinant histone H3 (a.a. 1-136(end)) with K27C, C97A, and C111A mutations, and an N-terminal His-tag, expressed in an E. coli expression system. GenBank Accession No. NM_003532. The protein has been alkylated to produce the lysine analog, trimethyl aminoethylcysteine. MW = 16 kDa.
Description: Treponema Pallidum p47 (Syphilis) full length, recombinant protein from E. coli, fused with Beta-galactosidase (114 kDa) at the N-terminus, 1.00 mg/mL.
Recombinant Treponema Pallidum p17 Protein (Full Length)
Description: Treponema Pallidum p17 (Syphilis) full length, recombinant protein from E. coli, fused with Beta-galactosidase (114 kDa) at the N-terminus, 1.00 mg/mL.
Recombinant Treponema Pallidum p15 Protein (Full Length)
Description: Treponema Pallidum p15 (Syphilis) full length, recombinant protein from E. coli, fused with Beta-galactosidase (114 kDa) at the N-terminus, 1.00 mg/mL.
Description: The Homogeneous full length SHP-2 Assay Kit is a complete assay system designed to measure full length SHP-2 activity for screening and profiling applications. Using this kit, only two simple steps on a microtiter plate are needed to analyze the full length SHP-2 activity level. At the first step, the SHP-2 enzyme is preincubated with SHP-2 Activating Peptide to activate the enzyme. At the second step, the fluorogenic substrate, DiFMUP, is added in the mixture and the enzymatic activity releases DiFMU fluorophore that can then be measured using a fluorescence reader.
Description: Human recombinant histone tetramer, full length, consisting of 2 molecules each of histones H3 (a.a. 1-136(end)), and H4 (a.a. 1-103(end)), each with an N-terminal His-tag, expressed in an E. coli expression system. GenBank Accession Nos. are NM_003532, and NM_003548, respectively. Global MW = 57 kDa.
Description: Human recombinant histone tetramer, full length, consisting of 2 molecules each of histones H3 (a.a. 1-136(end)), and H4 (a.a. 1-103(end)), each with an N-terminal His-tag, expressed in an E. coli expression system. GenBank Accession Nos. are NM_003532, and NM_003548, respectively. Global MW = 57 kDa.
Description: Treponema Pallidum Treponemal Membrane Protein A (TmpA) Full Length, recombinant protein from E. coli, fused with Beta-galactosidase (114 kDa) at the N-terminus, MW 42 kDa, 1.00 mg/mL.
SARS CoV-2 full length spike protein nanodisc complex
Description: The coronavirus, also known as SARS-CoV-2, enters the cell by using its surface SPIKE. SPIKE is processed on the cell's surface by TMPRSS2, a serine protease. It then subsequently binds to ACE2 a cell surface receptor. The Native SPIKE protein is a trimer that is located in the coronavirus membrane. Therefore to get pure & native SPIKE the trimer needs to be kept intact. Our lab staff achieved this in three different ways: MSP nanodiscs, based on MSP proteins Detergent Mycelles, as you can see here Synthetic nanodiscs
Description: Biotinylated Human Histone 2A, GenBank Accession No. NM_033445, a.a. 2-130(end) with N-terminal His-tag and C-terminal Cys. MW = 15 kDa, expressed in an E. coli expression system.
Description: Biotinylated Human Histone 2A, GenBank Accession No. NM_033445, a.a. 2-130(end) with N-terminal His-tag and C-terminal Cys. MW = 15 kDa, expressed in an E. coli expression system.
Description: The coronavirus, also known as SARS-CoV-2, enters the cell by using its surface SPIKE. SPIKE is processed on the cell's surface by TMPRSS2, a serine protease. It then subsequently binds to ACE2 a cell surface receptor. The Native SPIKE protein is a trimer that is located in the coronavirus membrane. Therefore to get pure & native SPIKE the trimer needs to be kept intact. Our lab staff achieved this in three different ways: MSP nanodiscs, based on MSP proteins Detergent Mycelles, as you can see here Synthetic nanodiscs
SARS CoV-2 full length spike protein in DIBMA Glycerol
Description: The coronavirus, also known as SARS-CoV-2, enters the cell by using its surface SPIKE. SPIKE is processed on the cell's surface by TMPRSS2, a serine protease. It then subsequently binds to ACE2 a cell surface receptor. The Native SPIKE protein is a trimer that is located in the coronavirus membrane. Therefore to get pure & native SPIKE the trimer needs to be kept intact. Our lab staff achieved this in three different ways: MSP nanodiscs, based on MSP proteins Detergent Mycelles, as you can see here Synthetic nanodiscs
Human Oxyntomodulin (OXM) peptide full length (37-aa)
Synthesis of Full-Size cDNA Infectious Clones of Soybean Mosaic Virus and Purposeful Identification of a Key Amino Acid within the Silencing Suppressor Hc-Professional
Soybean mosaic virus (SMV), which belongs to the Potyviridae, causes vital reductions in soybean yield and seed high quality. On this examine, each tag-free and reporter gene inexperienced fluorescent protein (GFP)-containing infectious clones for the SMV N1 pressure have been constructed by Gibson meeting and with the yeast homologous recombination system, respectively.
Each infectious clones are appropriate for agroinfiltration on the mannequin host benthamiana and present robust infectivity for the pure host soybean and several other different legume species. Each infectious clones have been seed transmitted and prompted typical virus signs on seeds and progeny crops. We used the SMV-GFP infectious clone to additional examine the function of key amino acids within the silencing suppressor helper component-proteinase (Hc-Professional).
Amongst twelve amino acid substitution mutants, the co-expression of mutant 2-with an Asparagine→Leucine substitution at place 182 of the FRNK (Phe-Arg-Asn-Lys) motif-attenuated viral signs and alleviated the host progress retardation brought on by SMV.
Furthermore, the Hc-Prom2 mutant confirmed stronger oligomerization than wild-type Hc-Professional. Taken collectively, the SMV infectious clones can be helpful for research of host-SMV interactions and useful gene characterization in soybeans and associated legume species, particularly by way of seed transmission properties. Moreover, the SMV-GFP infectious clone will even facilitate useful research of each virus and host genes in an benthamiana transient expression system.
Profiling of rice Cd-tolerant genes by way of yeast-based cDNA library survival screening
The bioaccumulation of cadmium (Cd) in crop and the next meals chain has aroused intensive considerations. Nevertheless, the underlying molecular mechanisms of plant Cd tolerance stay to be clarified from the viewpoint of novel candidate genes.
Right here we described a extremely environment friendly strategy for preliminary figuring out rice Cd-tolerant genes by way of the yeast-based cDNA library survival screening mixed with high-throughput sequencing technique. About 690 gene isoforms have been recognized as being Cd-tolerant candidates utilizing this shotgun strategy.
Among the many Cd-tolerant genes recognized, a number of classes of genes reminiscent of BAX inhibitor (BI), NAC transcription elements and Fast ALkalinization Elements (RALFs) have been of specific curiosity, and their perform of Cd tolerance was additional validated through heterologous expression, which urged that SNAC1, RALF12, OsBI-1 can confer Cd tolerance in yeast and tobacco leaves.
Relating to the genes concerned in ion transport, the validated Cd-tolerant heavy metal-associated area (HMAD) isoprenylated protein HIPP42 was significantly noteworthy. Additional elucidation of those genes related to Cd tolerance in rice will profit agricultural actions.
Single-Cell Transcriptomics of Immune Cells: Cell Isolation and cDNA Library Era for scRNA-Seq
Single-cell RNA-sequencing (scRNA-seq) allows a complete evaluation of the transcriptome of particular person cells by next-generation sequencing. ScRNA-seq gives an unbiased strategy to analyze the mobile heterogeneity and dynamics of various organic methods, together with the immune system. Optimization of the technical procedures carried out previous to RNA-seq evaluation is crucial to the success of a scRNA-seq experiment.
Right here, three main experimental procedures are described: (1) the isolation of immune CD8a+ T cells from main murine tissue, (2) the technology of single-cell cDNA libraries utilizing the 10× Genomics Chromium Controller and the Chromium Single Cell 3′ Answer, and (3) cDNA library high quality management. On this protocol, CD8a+ T cells are remoted from murine spleen tissue, however any cell kind of curiosity could be enriched and used for single-cell cDNA library technology and subsequent RNA-seq experiments.
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Human Chemokine C-C-Motif Receptor 6 (CCR6) in tissue homogenates, cell lysates and other biological fluids.
Human Chemokine C-C-Motif Receptor 6 (CCR6) ELISA Kit
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Human Chemokine C-C-Motif Receptor 6 (CCR6) in tissue homogenates, cell lysates and other biological fluids.
Human Chemokine C-C-Motif Receptor 6 (CCR6) ELISA Kit
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Human Chemokine C-C-Motif Receptor 6 (CCR6) in tissue homogenates, cell lysates and other biological fluids.
Human Chemokine C-C-Motif Receptor 6 (CCR6) ELISA Kit
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Human Chemokine C-C-Motif Receptor 6 (CCR6) in tissue homogenates, cell lysates and other biological fluids.
Human Chemokine C-C-Motif Receptor 6 (CCR6) ELISA Kit
Description: Enzyme-linked immunosorbent assay based on the Double-antibody Sandwich method for detection of Human Chemokine C-C-Motif Receptor 6 (CCR6) in samples from tissue homogenates, cell lysates and other biological fluids with no significant corss-reactivity with analogues from other species.
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Mouse Chemokine C-C-Motif Receptor 6 (CCR6) in Tissue homogenates, cell lysates and other biological fluids.
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Mouse Chemokine C-C-Motif Receptor 6 (CCR6) in Tissue homogenates, cell lysates and other biological fluids.
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Mouse Chemokine C-C-Motif Receptor 6 (CCR6) in Tissue homogenates, cell lysates and other biological fluids.
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Mouse Chemokine C-C-Motif Receptor 6 (CCR6) in Tissue homogenates, cell lysates and other biological fluids.
Description: Enzyme-linked immunosorbent assay based on the Double-antibody Sandwich method for detection of Mouse Chemokine C-C-Motif Receptor 6 (CCR6) in samples from Tissue homogenates, cell lysates and other biological fluids. with no significant corss-reactivity with analogues from other species.
Description: A polyclonal antibody against CCR6. Recognizes CCR6 from Human. This antibody is Unconjugated. Tested in the following application: ELISA, WB; Recommended dilution: WB:1:500-1:2000
Description: A polyclonal antibody against CCR6. Recognizes CCR6 from Human. This antibody is Unconjugated. Tested in the following application: ELISA, IHC;ELISA:1:1000-1:5000, IHC:1:50-1:200
Description: A polyclonal antibody against CCR6. Recognizes CCR6 from Human. This antibody is Unconjugated. Tested in the following application: ELISA, WB, IHC;ELISA:1:2000-1:10000, WB:1:1000-1:5000, IHC:1:50-1:200
Description: CCR6, also termed CKRL3, encodes a 369-amino acid polypeptide with greatest similarity to the family of alpha-chemokine-binding receptors. Unlike most chemokine receptor genes, it is encoded by more than 1 exon. Weakly expressed as a 4-kb transcript in spleen, lymph nodes, peripheral blood lymphocytes and appendix, CCR6 gene is located at 6q27. As the receptor for MIP-3-alpha, its activation leads to phospholipase C-dependent intracellular Ca(2+) mobilization. Additionally, CCR6 are markedly upregulated in psoriasis.
Description: A Monoclonal antibody against Human CCR7/CKR7. The antibodies are raised in Rabbit and are from clone E75. This antibody is applicable in WB, FC
