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Cold Spring Harbor Protocols

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https://read.qxmd.com/read/30606753/skin-grafting-in-xenopus-laevis-a-technique-for-assessing-development-and-immunological-disparity
#1
Yumi Izutsu
Skin grafting in the amphibian Xenopus laevis has been used to detect not only allogeneic antigens that differ by minor H antigens or by one MHC haplotype, but also to detect ontogeny-specific antigens (including both emerging adult- and disappearing larval-specific) during metamorphosis. To understand the mechanisms underlying allogeneic tolerance or immune responses against larval- and/or adult-specific antigens, a complete MHC homozygous, inbred strain is the most appropriate experimental model. The inbred J strain established in Japan is used here...
January 3, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30606752/mapping-chromatin-features-of-xenopus-embryos
#2
George E Gentsch, James C Smith
Chromatin immunoprecipitation (ChIP) combined with genomic analysis provides a global snapshot of protein-DNA interactions in the context of chromatin, yielding insights into which genome loci might be regulated by the DNA-associated protein under investigation. This protocol is an update of a previous version and describes how to perform ChIP on intact or dissected Xenopus embryos. The ChIP-isolated DNA fragments are suitable for both deep sequencing (ChIP-Seq) and quantitative polymerase chain reaction (ChIP-qPCR)...
January 3, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30824627/lipofection
#3
Priti Kumar, Arvindhan Nagarajan, Pradeep D Uchil
In the past, cloned DNA was introduced into cultured eukaryotic cells chiefly by biochemical methods using either calcium phosphate or diethylaminoethyl (DEAE)-dextran. Lipid reagents are now preferred because of the high efficiency of transfection that can be obtained and because of the ability of this class of reagents to mediate transfection of all types of nucleic acids into a wide range of cell types.
March 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30824626/measurement-of-radioactivity-in-nucleic-acids
#4
Michael R Green, Joseph Sambrook
Radioactive isotopes are used as tracers to monitor the progress of many reactions used to synthesize DNA and RNA. To calculate the efficiency of such reactions, it is necessary to measure accurately the proportion of the radioactive precursor incorporated into the desired product. An effective method to achieve this goal is differential precipitation of the nucleic acid products with trichloroacetic acid (TCA).
March 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30824625/spun-column-chromatography
#5
Michael R Green, Joseph Sambrook
This protocol describes the setup and use of Sephadex G-50 or Bio-Gel columns for the purification of DNA.
March 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30824624/random-priming-labeling-of-purified-dna-fragments-by-extension-of-random-oligonucleotides
#6
Michael R Green, Joseph Sambrook
In this protocol, oligonucleotides that are heterogeneous in sequence serve as primers for the initiation of DNA synthesis on single-stranded templates. Labeled dNTPs ([α-32 P]dNTPs or biotin-, DIG-, or fluorescein-labeled dUTPs) are incorporated into the new DNA by the Klenow fragment of Escherichia coli DNA polymerase I (Pol I). The method can be adapted to radiolabel DNA in slices cut from gels cast with low-melting-temperature agarose.
March 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30824623/transfected-dendritic-cell-immunizations
#7
Edward A Greenfield, James DeCaprio, Mohan Brahmandam
Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs). An efficient way of generating antibodies is to introduce antigens of interest into DCs and then inject them into the host. This will result in initiation of an antigen-specific immune response mediated by T-cell immunity. Apoptosis of DCs expressing transgenic proteins results in enhanced immunity through cross-presentation by endogenous DCs in vivo. The major advantage of this technology is prolonged presentation of antigens that are synthesized endogenously and their presentation by both modified DCs as well as endogenous DCs to the immune system of the host...
March 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30824622/labeling-antibodies-using-a-maleimido-dye
#8
Eric A Berg, Jordan B Fishman
Fluorophore-maleimide derivatives are effective for labeling sulfhydryl-containing molecules. Maleimide groups react with free thiols at pH 6.5-7.5 forming a covalent bond. Reducing agents should be avoided during the conjugation step. This protocol uses the cross-linker N -succinimidyl S -acetylthioacetate (SATA) to introduce thiol groups on the antibody while maintaining the divalent nature of the antibody. Alternatively, the antibody can be digested and reduced to monovalent Fab fragments, which can then be labeled directly with maleimido derivatives...
March 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30824621/labeling-antibodies-using-n-hydroxysuccinimide-nhs-fluorescein
#9
Eric A Berg, Jordan B Fishman
N -Hydroxysuccinimide (NHS)-ester derivatives are among the most commonly used reagents for labeling proteins. The method described here can be adapted to use practically any NHS fluorophore. Generally, a fluorophore is covalently bound to a macromolecule such as an antibody and acts as a reporter molecule used to measure the presence of the macromolecule. These fluorescently labeled bioactive reagents are suitable for use in immunofluorescence, flow cytometry, and numerous other biological applications. There are several widely used dyes available in convenient formats...
March 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30824620/rnai-in-drosophila-s2-cells-by-dsrna-soaking
#10
Chengjian Li, Phillip D Zamore
This is a simple method for inducing RNAi in Drosophila S2 cells by soaking the cells in medium containing dsRNAs. In comparison with transfection, soaking requires fewer procedures and avoids the cost of transfection reagents, making it the method of choice for high-throughput RNAi screening. Moreover, soaking avoids potential toxicity associated with transfection reagents. However, delivery of dsRNA to S2 cells by soaking is less efficient than transfection, and genes whose expression has proved difficult to repress using soaked dsRNA often can be suppressed by transfecting the dsRNA multiple times...
March 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30824619/preparation-of-dsrnas-for-rnai-by-in-vitro-transcription
#11
Chengjian Li, Phillip D Zamore
This is a simple and widely used method for the preparation of dsRNA using a polymerase chain reaction (PCR) template-based, in vitro transcription reaction. The resulting dsRNA is then used to trigger RNAi in an appropriate cell or organism.
March 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30824618/histochemical-staining-of-cell-monolayers-for-%C3%AE-galactosidase
#12
Priti Kumar, Arvindhan Nagarajan, Pradeep D Uchil
There are several methods for assaying the success of transient transfections. If a plasmid expressing Escherichia coli β-galactosidase was used, then this histochemical staining procedure is simple to perform and yields dependable results. The following method was designed for cells growing in 60-mm culture dishes.
March 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30824617/dna-transfection-mediated-by-cationic-lipid-reagents
#13
Priti Kumar, Arvindhan Nagarajan, Pradeep D Uchil
Liposomal transfection reagents vary in their ability to transfect cell lines efficiently. Some are generalists, whereas others are best used with specific cell types. The nonliposomal FuGENE 6 and the cationic liposomal Lipofectamine 2000 are examples of reagents that can successfully transfect most adherent and suspension cell types (including several primary and hard-to-transfect cell types) with negligible toxicity and a minimal number of manipulations. Importantly, both reagents can be used to transfect cells in the presence of serum, minimizing the number of manipulations during the transfection procedure...
March 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30824616/long-and-accurate-polymerase-chain-reaction-la-pcr
#14
Michael R Green, Joseph Sambrook
The standard polymerase chain reaction (PCR) is easily capable of amplifying segments of DNA smaller than ∼3 kb in length-sufficient for most purposes, but not enough to amplify an entire mammalian gene, nor even a cDNA of average dimensions. Instead of full-length products, standard PCR amplification of longer templates generates variously sized truncated molecules that appear as unattractive smears on a gel. Long and accurate PCR (LA PCR) addresses the issue in part by using a mixture of two different thermostable DNA polymerases to catalyze the amplification reaction...
March 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30824615/preparation-of-polymerase-chain-reaction-templates-from-embryonic-and-adult-mouse-tissue-samples
#15
Janet Rossant, Andras Nagy
A simple cell or tissue lysate can provide a sufficient quality and amount of template DNA for polymerase chain reaction (PCR). This protocol can be used to prepare embryonic tissues, yolk sac, ear punch, toe, or 1- to 2-mm (maximum 5 mm) tail samples for PCR analysis.
March 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30710029/how-to-win-the-battle-with-rnase
#16
Michael R Green, Joseph Sambrook
Because ribose residues carry hydroxyl groups in both the 2' and 3' positions, RNA is chemically much more reactive than DNA and is easy prey to cleavage by contaminating RNases-enzymes with various specificities that share the property of hydrolyzing diester bonds linking phosphate and ribose residues. Because RNases are released from cells following lysis and are present on the skin, constant vigilance is required to prevent contamination of glassware and bench tops and the creation of aerosols carrying RNase...
February 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30710028/isolation-of-dna-fragments-from-polyacrylamide-gels-by-the-crush-and-soak-method
#17
Michael R Green, Joseph Sambrook
The standard method to recover fragments of DNA from polyacrylamide gels is the "crush and soak" technique. The eluted DNA is generally free of contaminants that inhibit enzymes or that are toxic to transfected or microinjected cells. The method requires time but little labor and results in recovery of <30%-90%, depending on the size of the DNA fragment. It can be used to isolate both double-stranded and single-stranded DNAs from neutral and denaturing polyacrylamide gels, respectively. The method is widely used to isolate synthetic oligonucleotides from denaturing polyacrylamide gels...
February 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30710027/tandem-immunoaffinity-purification-using-anti-flag-and-anti-ha-antibodies
#18
James DeCaprio, Thomas O Kohl
The immunoaffinity purification of target proteins followed by the identification and characterization of associated proteins by mass spectrometry is a widely used technique. An immunoaffinity purification bears resemblance to a standard immunoprecipitation; however, the end product for mass spectrometric analysis in the femtomole (10-15 ) to attomole (10-18 ) range is required to be of exceptional purity. This high degree of sensitivity in detection renders it of extreme importance to eliminate most if not all of the nonspecific background proteins and can be achieved by performing a tandem affinity purification (TAP)...
February 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30710026/cross-linking-antibodies-to-beads-with-disuccinimidyl-suberate-dss
#19
James DeCaprio, Thomas O Kohl
This protocol describes the cross-linking of antibodies to either Protein A or G agarose beads using disuccinimidyl suberate (DSS), a bifunctional cross-linker capable of directly reacting with two different amines to form stable amide bonds. Proteins, including antibodies, generally display several primary amines in the side chains of lysine (K) residues and the amino terminus of each polypeptide that represent available potential targets for N -hydroxysuccinimide (NHS)-ester cross-linking reagents. The antibody-bead cross-linking process generates a reusable resource of antibody and beads, commonly referred to as an antibody-specific resin, and can be repeatedly used for the immunoprecipitation of specific proteins if treated and stored correctly...
February 1, 2019: Cold Spring Harbor Protocols
https://read.qxmd.com/read/30710025/cross-linking-antibodies-to-beads-using-dimethyl-pimelimidate-dmp
#20
James DeCaprio, Thomas O Kohl
This protocol describes the cross-linking of antibodies to either Protein A or G agarose beads using dimethyl pimelimidate (DMP). DMP contains an imidoester at each end of a 7-carbon spacer arm and forms an amidine bond with amino groups at alkaline pH; however, cross-linking is more efficient when performed at pH >8. DMP will react with primary amines; thus, it is important that the cross-linking procedure is conducted using nonamine-containing buffers. Following the antibody-bead incubation, beads are washed in Borate buffer to remove residual amines from the Tris buffer...
February 1, 2019: Cold Spring Harbor Protocols
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