Millipore Sigma Vibrant Logo
Attention: We have moved. Merck Millipore products are no longer available for purchase on MerckMillipore.com.Learn More
 

choloroform


43 Results Advanced Search  
Showing
Products (14)
Documents (14)
Site Content (15)

Narrow Your Results Use the filters below to refine your search

Document Type

  • (11)
  • (2)
  • (1)

Application Type

  • (2)

Field of Activity

  • (2)

Sample

  • (1)
  • (1)
Can't Find What You're Looking For?
Contact Customer Service

 

  • Expression and induction of cytochrome p450 isoenzymes in human skin equivalents. 20090406

    Organotypic skin models are frequently used for a wide range of applications and latterly also for dermatotoxicological studies. To evaluate their practicability for the investigation of xenobiotic metabolism in human skin we compared three types of organotypic skin models, acquired by purchase from different manufacturers, to a self-constructed in-house model with regard to cytochrome P450 (CYP) isoenzyme expression on mRNA and protein level and the inducibility of these enzymes by aryl hydrocarbon receptor ligands. To induce enzyme activity, models were treated with benzanthracene, liquor carbonis detergens, pix lithanthracis or dimethyl sulfoxide as a solvent control. RNA was isolated by phenol-chloroform extraction and purified. Gene expression patterns were studied by cDNA microarray analysis. Microarray data were confirmed by real-time PCR. For quality control of the models and to detect and localize enzyme expression, immunofluorescence staining was performed with antibodies against CYPs and structure proteins. The immunofluorescence staining demonstrated the regular structure of our models. We could provide evidence for the expression of CYP types 1A1, 1B1, 2E1, 2C and 3A5 in organotypic skin models. The expression of CYP1A1 and CYP1B1 was highly inducible by treatment with liquor carbonis detergens. The proof of the expression and inducibility of CYP enzymes in organotypic skin models suggests that skin equivalents are a valuable tool that can emulate CYP-dependent metabolism of drugs and other xenobiotics in human skin.
    Document Type:
    Reference
    Product Catalog Number:
    AB1254
    Product Catalog Name:
    Anti-Cytochrome P450 Enzyme CYP3A4 Antibody

  • Chromatin immunoprecipitation (ChIP): revisiting the efficacy of sample preparation, sonication, quantification of sheared DNA, and analysis via PCR. 22046253

    The "quantitative" ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR. We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry. However, DNA extracted from the Mock-ChIP supernatant via the phenol-chloroform-isoamyl alcohol (PCIA) method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay. This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR (rtPCR). Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA. The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Chemoprevention and inhibition of P-glycoprotein in cancer cells by Chinese medicinal herbs. 18690658

    Many of the herbal extracts used in the Chinese clinical medical routine inhibit the growth of tumor cells. In the present work, extracts of 12 selected herbs were prepared with methanol, chloroform, ethyl acetate and water, and the effects of these on the multidrug resistance (MDR) and P-glycoprotein of mouse lymphoma cells transfected with the human mdr1 gene and on a human lung alveolar epithelial cell line were investigated. The extracts were tested for antiproliferative effects, and the reversal of MDR in mouse lymphoma cells. The possible chemopreventive effect of the chloroform extracts was studied on the expression of cytomegalovirus (CMV) immediate-early (IE) antigen in human lung cancer cells (A549). The antimicrobial effects of the extracts were tested on some representative micro-organisms. Certain of the chloroform extracts of the plant materials were the most effective compounds on the reversal of MDR. Two of the chloroform extracts enhanced the antiproliferative effect of doxorubicin on MDR mouse lymphoma cells. The selected extracts did not show any antibacterial effect with the agar diffusion method. Certain chloroform extracts decreased the intermediate IE antigen expression of CMV in A459 cells. Copyright (c) 2008 John Wiley Sons, Ltd.
    Document Type:
    Reference
    Product Catalog Number:
    MAB810

  • Isolation and partial characterization of neurofibrillary tangles and amyloid plaque core in Alzheimer's disease: immunohistological studies. 3772397

    Fractions enriched in neurofibrillary tangles (NFT) and amyloid fibrils were isolated from the cerebral cortex of three cases of senile dementia of the Alzheimer type. Distilled water suspensions of these fractions were excluded from all pore size gels and resisted digestion with various proteolytic enzymes. Formic acid/chloroform treatment of each fraction resulted in the appearance of 4,000-6,000, 15,000-17,000 and 24,000 molecular weight proteins, with concomitant diminution in the amount of excluded material at the top of each gel. The 4,000-6,000 dalton band was best seen in fractions containing randomly arranged amyloid fibrils, and its amino acid composition resembled that of the recently reported "beta" protein. A polyclonal antiserum to purified NFT reacted with tangles in neurons and in dystrophic neurites around plaques by immunoperoxidase staining. No reaction was obtained with cerebrovascular or plaque core amyloid immunohistologically, or with the 4-6 kD protein on immunoblots. Cross-reactivity with the neurofibrillary lesions occurring in Pick's disease, progressive supranuclear palsy, postencephalitic Parkinsonism and dementia pugilistica was also seen. Specific binding of this antiserum to the double filamentous structure was confirmed by immunoelectron microscopy. Although the presence of "beta" protein in both NFT and amyloid-containing fractions suggests that it may be an important constituent of both, cross-contamination cannot be excluded.
    Document Type:
    Reference
    Product Catalog Number:
    AB1518
    Product Catalog Name:
    Anti-Neurofibrillary Tangles Antibody

  • Creation of highly aligned electrospun poly-L-lactic acid fibers for nerve regeneration applications. 19104139

    Aligned, electrospun polymer fibers have shown considerable promise in directing regenerating axons in vitro and in vivo. However, in several studies, final electrospinning parameters are presented for producing aligned fiber scaffolds, and alignment where minimal fiber crossing occurs is not achieved. Highly aligned species are necessary for neural tissue engineering applications to ensure that axonal extension occurs through a regenerating environment efficiently. Axonal outgrowth on fibers that deviate from the natural axis of growth may delay axonal extension from one end of a scaffold to the other. Therefore, producing aligned fiber scaffolds with little fiber crossing is essential. In this study, the contributions of four electrospinning parameters (collection disk rotation speed, needle size, needle tip shape and syringe pump flow rate) were investigated thoroughly with the goal of finding parameters to obtain highly aligned electrospun fibers made from poly-L-lactic acid (PLLA). Using an 8 wt% PLLA solution in chloroform, a collection disk rotation speed of 1000 revolutions per minute (rpm), a 22 gauge, sharp-tip needle and a syringe pump rate of 2 ml h(-1) produced highly aligned fiber (1.2-1.6 microm in diameter) scaffolds verified using a fast Fourier transform and a fiber alignment quantification technique. Additionally, the application of an insulating sheath around the needle tip improved the rate of fiber deposition (electrospinning efficiency). Optimized scaffolds were then evaluated in vitro using embryonic stage nine (E9) chick dorsal root ganglia (DRGs) and rat Schwann cells (SCs). To demonstrate the importance of creating highly aligned scaffolds to direct neurite outgrowth, scaffolds were created that contained crossing fibers. Neurites on these scaffolds were directed down the axis of the aligned fibers, but neurites also grew along the crossed fibers. At times, these crossed fibers even stopped further axonal extension. Highly aligned PLLA fibers generated under optimized electrospinning conditions guided neurite and SC growth along the aligned fibers. Schwann cells demonstrated the bipolar phenotype seen along the fibers. Using a novel technique to determine fiber density, an increase in fiber density correlated to an increase in the number of neurites, but average neurite length was not statistically different between the two different fiber densities. Together, this work presents methods by which to produce highly aligned fiber scaffolds efficiently and techniques for assessing neurite outgrowth on different fiber scaffolds, while suggesting that crossing fibers may be detrimental in fostering efficient, directed axonal outgrowth.
    Document Type:
    Reference
    Product Catalog Number:
    05-100

  • Generation of p53 target database via integration of microarray and global p53 DNA-binding site analysis. 15220520

    The completion of the human genome sequence and availability of cDNA microarray technology provide new approaches to explore global cellular regulatory mechanisms. Here we present a strategy to identify genes regulated by specific transcription factors in the human genome, and apply it to p53. We first collected promoters or introns of all genes available using two methods: GenBank annotation and a computationally derived transcript map. The FindPatterns program is then used to search sequences in regulatory regions that match the p53 DNA-binding consensus sequence, resulting in the p53 Target Database. This database collects human genes that have at least one p53 DNA-binding sequence in their regulatory region. cDNA microarray was also used to identify genes that respond to p53 at a genomic scale. Integration of the microarray data and the p53 Target Database should greatly enrich direct p53 target genes. Taqman analysis and quantitative chromatin immunoprecipitation analysis are used to validate the in silico prediction and microarray data. Enrichment factor analysis is used to demonstrate that in silico prediction greatly enriches for genes that are transcriptionally regulated by p53 and assists us to identify other signaling pathways that are potentially connected to p53. The approaches can be extended to other transcription factors. The methods shown here illustrate a novel approach to the analysis of global gene regulatory networks through the integration of human genomic sequence information and genome-wide gene expression analysis.
    Document Type:
    Reference
    Product Catalog Number:
    16-157
    Product Catalog Name:
    Protein A Agarose/Salmon Sperm DNA, 2.5 mL

  • Chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. 21808222

    Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and often challenging. The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.
    Document Type:
    Reference
    Product Catalog Number:
    20-163
    Product Catalog Name:
    SDS Lysis Buffer - for use in ChIP Assay