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Microbial Mutagenicity Assay: Ames Test
Authors:  Urvashi Vijay, Sonal Gupta, Priyanka Mathur, Prashanth Suravajhala and Pradeep Bhatnagar, date: 03/20/2018, view: 1555, Q&A: 2
The Microbial mutagenicity Ames test is a bacterial bioassay accomplished in vitro to evaluate the mutagenicity of various environmental carcinogens and toxins. While Ames test is used to identify the revert mutations which are present in strains, it can also be used to detect the mutagenicity of environmental samples such as drugs, dyes, ...
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Ames Test
[Feedback 1] Dear Sir/Mam I have read your article in bio-protocol and found so many mistakes in the protocol. Please go through the composition of Vogel Bonner Medium Recipe 1 in your protocol. 1) All the constituents listed are in mg (miligram), but actually it is in grams. 2) the composition Agar mentioned in recipe 9, 10 and 11 in mg but actually it is in grams. 3) Please check all the compositions carefully. The Protocol mentioned here is not reproducible because article have so many composition error..

This protocol is a test page
Authors:  hypo 单海波 and Jianguo Ruan, date: 08/30/2017, view: 1501, Q&A: 39
TRIzol中苯酚和硫氰酸胍,从组织细胞中快速抽提总RNA,并在匀浆和裂解细胞时保持RNA的完整性。氯仿萃取有机相后,RNA溶于水中相中,用异丙醇沉淀RNA。
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Antibody Purification from Western Blotting
Author:  Lin Fang, date: 03/20/2012, view: 12145, Q&A: 2
This protocol describes a method of purifying antibodies from sera with denatured antigens immobilized on western blot membranes. Advantages include (1) fast and easy; (2) purification of antibody with antigen in denatured form allows high yield in case antigen protein solubility is limited. Disadvantage is that possible antibodies that recognize ...
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Purifying Commercial Antibodies (Not from Sera)

Invadopodia Detection and Gelatin Degradation Assay
Author:  Begoña Díaz, date: 12/20/2013, view: 11057, Q&A: 0
This protocol is designed to quantify invadopodia formation and activity. Invadopodia are protrusive structures elaborated by cancer cells that mediate cell attachment and remodeling of the extracellular matrix. These structures contribute to the ability of cancer cells to invade and metastasize. In this protocol, both the presence of invadopodia ...
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Invadopodia Detection and Gelatin Degradation Assay...this protocol is excellent !

Mouse CD8+ T Cell Migration in vitro and CXCR3 Internalization Assays
Authors:  Rosa Barreira da Silva and Matthew L. Albert, date: 03/20/2017, view: 3492, Q&A: 0
Chemokines are molecules that regulate the positioning of cells during homeostasis and inflammation. CXCL10 is an interferon-induced chemokine that attracts cells that express the chemokine receptor CXCR3 on their surface. CXCL10 expression is often induced upon inflammation and guides lymphocytes, such as T and NK cells, into the injured tissues. ...
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Great protocol!

Antisense Oligonucleotide-mediated Knockdown in Mammary Tumor Organoids
Authors:  Sarah D. Diermeier and David L. Spector, date: 08/20/2017, view: 2127, Q&A: 0
Primary mammary tumor organoids grown in 3D are an excellent system to study tumor biology. They resemble the organization and physiology of native epithelia more closely than cancer cell lines grown in 2D, and additionally model interactions with the ECM (Boj et al., 2015; Clevers, 2016; Shamir and Ewald, 2014). Mammary tumor organoids ...
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Antisense oligonucleotide treatment in mammalian cell

Plant Tissue Trypan Blue Staining During Phytopathogen Infection
In this protocol plant tissue is stained with trypan blue dye allowing the researcher to visualize cell death. Specifically this method avoids the use of the carcinogen compound chloral hydrate, making this classical method of staining safer and faster than ever. The protocol is applied specifically to detect cell death on Arabidopsis ...
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Mahmudul

Isolation of Human PBMCs
Authors:  Santosh K Panda and Balachandran Ravindran, date: 02/05/2013, view: 32934, Q&A: 2
Peripheral blood mononuclear cells (PBMCs) are chiefly lymphocytes and monocytes. PBMCs are separated from the whole blood by a density gradient centrifugation method using Ficoll Histopaque.
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Isolation of Human PBMCs

Isolation of Primary Human Skeletal Muscle Cells
Authors:  Janelle M. Spinazzola and Emanuela Gussoni, date: 11/05/2017, view: 1482, Q&A: 0
Primary myoblast culture is a valuable tool in research of muscle disease, pathophysiology, and pharmacology. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing ...
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Adherent-invasive Escherichia coli Biofilm Formation Assays
Authors:  Benoit Chassaing and Arlette Darfeuille-Michaud, date: 12/05/2013, view: 6441, Q&A: 0
Patients with Crohn’s disease are abnormally colonized by adherent-invasive Escherichia coli (AIEC) bacteria (Chassaing and Darfeuille-Michaud, 2011). These bacteria are able to adhere to and invade intestinal epithelial cells (IEC), to replicate within macrophages, and were recently described to be able to form biofilms (Martinez-Medina ...
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Dr

Clonogenic Assay
Author:  Xiaodong Yang, date: 05/20/2012, view: 54637, Q&A: 5
Clonogenic assays serve as a useful tool to test whether a given cancer therapy can reduce the clonogenic survival of tumor cells. A colony is defined as a cluster of at least 50 cells that can often only be determined microscopically. A clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing ...
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A Quick and Easy Method for Making Competent Escherichia coli Cells for Transformation Using Rubidium Chloride
Authors:  Nidhi Sharma, M. Ximena Anleu Gil and Diego Wengier, date: 11/05/2017, view: 2064, Q&A: 0
This protocol describes a quick and efficient method to make competent E. coli cells for transformation using rubidium chloride. Commercial competent cells are expensive and this protocol provides a cheaper alternative to them.
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Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions
Authors:  Amanda Hopes, Vladimir Nekrasov, Nigel Belshaw, Irina Grouneva, Sophien Kamoun and Thomas Mock, date: 12/05/2017, view: 1972, Q&A: 1
Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include ...
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MTT Assay for Cytotoxicity Assessment in Oryza sativa Root Tissue
Authors:  Snehalata Majumdar, Titir Guha and Rita Kundu, date: 11/20/2017, view: 1488, Q&A: 0
Cytotoxicity of different compounds are commonly evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This assay is mainly used to study cell viability in cell lines (Carmichael et al., 1987). In this study, the protocol is being used to determine the cell viability of plant roots, treated with different ...
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Measurement of Endogenous H2O2 and NO and Cell Viability by Confocal Laser Scanning Microscopy
Authors:  Mi-Mi Wu, Xian-Ge Ma and Jun-Min He, date: 10/05/2013, view: 7152, Q&A: 1
Recently, there is compelling evidence that hydrogen peroxide (H2O2) and nitric oxide (NO) function as signaling molecules in plants, mediating a range of responses including stomatal movement. Thus, the choice of sensitive methods for detection of endogenous H2O2 and NO in guard cells are very important ...
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Extraction and Purification of Mycobacterial Mycolic Acids
Authors:  Christian M. Dupont and Laurent Kremer, date: 10/20/2014, view: 6268, Q&A: 0
Mycolic acids are major long-chain fatty acids, containing up to 80-90 carbon atoms that represent essential components of the mycobacterial cell wall (Pawelczyk and Kremer, 2014). Each mycobacterial species possesses a specific mycolic acid profile characterized by various chemical modifications that decorate the lipid. Mycolic acids play a ...
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Worked like a charm!

Laser Scanning Confocal Microcopy for Arabidopsis Epidermal, Mesophyll, and Vascular Parenchyma Cells
Authors:  Christian Elowsky, Yashitola Wamboldt and Sally Mackenzie, date: 03/05/2017, view: 3359, Q&A: 0
Investigation of protein targeting to plastids in plants by confocal laser scanning microscopy (CLSM) can be complicated by numerous sources of artifact, ranging from misinterpretations from in vivo protein over-expression, false fluorescence in cells under stress, and organellar mis-identification. Our studies have focused on the ...
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E. coli Genomic DNA Extraction
Author:  Fanglian He, date: 07/20/2011, view: 119947, Q&A: 49
This protocol uses phenol/chloroform method to purify genomic DNA without using commercial kits.
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Helpful

Minimum Inhibitory Concentration (MIC) Assay for Antifungal Drugs
Authors:  Jinglin L. Xie, Sheena D. Singh-Babak and Leah E. Cowen, date: 10/20/2012, view: 13994, Q&A: 0
The Minimum Inhibitory Concentration (MIC) Assay is widely used to measure the susceptibility of yeasts to antifungal agents. In serial two-fold dilutions, the lowest concentration of antifungal drug that is sufficient to inhibit fungal growth is the MIC. Typically, 50% inhibitory (MIC50) or 80% inhibitory (MIC80) values are ...
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Validation of protocol for antifungal MIC assay

Dimethylmethylene Blue Assay (DMMB)
Authors:  Vivien Jane Coulson- Thomas and Tarsis Ferreira Gesteira, date: 09/20/2014, view: 26726, Q&A: 6
Glycosaminoglycans (GAGs) are long unbranched polysaccharides consisting of repeating disaccharide units composed of a hexosamine (glucosamine or galactosamine) and a hexuronic acid (glucuronic or iduronic acid). Depending on the disaccharide unit the GAGs can be organized into five groups: chondroitin sulfate, dermatan sulfate, heparan sulfate, ...
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Fractionation by Ultracentrifugation of Gram Negative Cytoplasmic and Membrane Proteins
Authors:  Sara M Sandrini, Richard Haigh and Primrose P. E. Freestone, date: 11/05/2014, view: 9299, Q&A: 1
Protein fractionation is a useful separation process which divides membrane proteins (including those located in the outer and inner membrane) and cytoplasmic proteins into discrete fractions. Fractionation of proteins can simplify analysis of the numbers of proteins present, and therefore make easier to characterize any environmentally or ...
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Immunofluorescent Staining of Mouse Intestinal Stem Cells
Authors:  Kevin P. O’Rourke, Lukas E Dow and Scott W Lowe, date: 02/20/2016, view: 12668, Q&A: 0
Immunofluorescent staining of organoids can be performed to visualize molecular markers of cell behavior. For example, cell proliferation marked by incorporation of nucleotide (EdU), or to observe markers of intestinal differentiation including paneth cells, goblet cells, or enterocytes (see Figure 1). In this protocol we detail a method to fix, ...
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Dr

Hydrogen Peroxide Measurement in Arabidopsis Root Tissue Using Amplex Red
Authors:  Tzvetina Brumbarova, Cham Thi Tuyet Le and Petra Bauer, date: 11/05/2016, view: 4713, Q&A: 0
This protocol describes the measurement of hydrogen peroxide (H2O2) content in Arabidopsis root tissue by using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. When root tissue is disrupted and resuspended in phosphate buffer, H2O2 is released from the cells. The obtained root ...
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Isolation and Growth of Adult Mouse Dorsal Root Ganglia Neurons
Authors:  Seong-il Lee and Joel Levine, date: 09/20/2015, view: 11103, Q&A: 0
Adult dorsal root ganglia neurons are among the few adult neuronal cell types that can be purified and grown relatively easily in dissociated cell culture. Here we describe a procedure for the isolation and growth of dissociated adult mouse DRG neurons using Percoll gradients and a chemically defined medium. These cultures can be used for ...
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Histostaining for Tissue Expression Pattern of Promoter-driven GUS Activity in Arabidopsis
Author:  Xiyan Li, date: 07/05/2011, view: 19462, Q&A: 3
Promoter-driven GUS (beta-glucuronidase) activity is the most commonly used technique for tissue-specific expression patterns in Arabidopsis. In this procedure, GUS enzyme converts 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) to a blue product. The staining is very sensitive. Processed samples can be examined under dissecting microscope or ...
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It worked but I had to optimize it with several modifications.

Pollen Fertility/viability Assay Using FDA Staining
Author:  Xiyan Li, date: 05/20/2011, view: 20477, Q&A: 2
Pollen grains can be fertile or sterile by nature. This method stains pollen grains for an enzyme as the vital indicator of membrane integrity. Only fertile grains fluoresce under microscopic examination.
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Very straightforward!

Extraction of Chloroplast Proteins from Transiently Transformed Nicotiana benthamiana Leaves
Author:  Joern Klinkenberg, date: 09/20/2014, view: 8829, Q&A: 0
This rapid protocol allows the extraction of chloroplast enriched proteins from Nicotiana benthamiana (N. benthamiana) leaves that were transiently transformed to express an epitope tagged protein of interest. Thus, it can serve as a tool to study the chloroplastidic localization of the protein of interest when it is combined ...
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Wnt Reporter Activity Assay
Author:  Chen Zhao, date: 07/20/2014, view: 14167, Q&A: 1
This protocol is for testing responses of a candidate cell line/cell lines to Wnt ligands or Wnt pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Wnt pathway responses. Canonical Wnt signaling activity transcriptionally induces Wnt ...
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Estimation of Wound Tissue Neutrophil and Macrophage Accumulation by Measuring Myeloperoxidase (MPO) and N-Acetyl-β-D-glucosaminidase (NAG) Activities
The inflammatory response is essential to the reestablishment of cutaneous homeostasis following injury. In this context, leukocytes arrive at the wound site and orchestrate essential events in the wound healing process. Therefore, the quantification of specific subsets of inflammatory cells in the wound tissue is of considerable interest. The ...
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MPO on small tissue biopsies

γ-Secretase Epsilon-cleavage Assay
γ-Secretase epsilon-cleavage assay is derived from the cell-based Tango assay (Kang et al., 2015), and is a fast and sensitive method to determine the initial cleavage of C99 by γ-secretase. In this protocol, we use HTL cells, which are HEK293 cells with a stably integrated luciferase reporter under the control of the bacterial tetO ...
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Simple Digital Photography for Assessing Biomass and Leaf Area Index in Cereals
Authors:  Jaume Casadesús and Dolors Villegas, date: 06/05/2015, view: 4938, Q&A: 0
These instructions refer to obtaining fast and low-labour estimates of ground cover, leaf area index and green biomass for a large number of plots, as those encountered in cereal breeding programs. The procedure includes obtaining the pictures in the field and processing them once downloaded to a computer.
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Zymogram Assay for the Detection of Peptidoglycan Hydrolases in Streptococcus mutans
Authors:  Delphine Dufour and Céline M. Lévesque, date: 08/20/2013, view: 7050, Q&A: 1
Peptidoglycan hydrolases or autolysins are enzymes capable of cleaving covalent bonds in bacterial peptidoglycan cell wall layer. They can participate in the cell division process, in the release of turnover products from peptidoglycan during cell growth, and in cell autolysis induced under particular conditions. The protocol for zymogram ...
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Zymogram tweaking

Chemiluminescence Detection of the Oxidative Burst in Plant Leaf Pieces
Authors:  Markus Albert, Melinka A. Butenko, Reidunn B. Aalen, Georg Felix and Mari Wildhagen, date: 03/20/2015, view: 7291, Q&A: 1
The production of 'reactive oxygen species' (ROS), also termed oxidative burst, is a typical cellular response of animals and plants to diverse biotic and abiotic stresses. Here, we outline the detection of the ROS-burst in plant leaf pieces using a luminol-based bioassay which allows for the detection of chemiluminescence. The assay was ...
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Gene Networks Based on the Graphical Gaussian Model
Author:  Shisong Ma, date: 02/20/2012, view: 8379, Q&A: 1
This protocol describes how to build a gene network based on the graphical Gaussian model (GGM) from large scale microarray data. GGM uses partial correlation coefficient (pcor) to infer co-expression relationship between genes. Compared to the traditional Pearson’ correlation coefficient, partial correlation is a better measurement of direct ...
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Very good one!

Optical Clearing Using SeeDB
Authors:  Meng-Tsen Ke, Satoshi Fujimoto and Takeshi Imai, date: 02/05/2014, view: 14214, Q&A: 1
We describe a water-based optical clearing agent, SeeDB (See Deep Brain), which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. SeeDB is a saturated solution of fructose (80.2% w/w) in water with 0.5% α-thioglycerol. In standard SeeDB optical ...
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Testing protocol on random adult mouse brain tissue section

Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves
Authors:  Arsalan Daudi and Jose A. O’Brien, date: 09/20/2012, view: 43517, Q&A: 19
In this protocol, the in situ detection of hydrogen peroxide (one of several reactive oxygen species) is described in mature Arabidopsis rosette leaves by staining with 3,3'-diaminobenzidine (DAB) using an adaptation of previous methods (Thordal-Christensen et al., 1997; Bindschedler et al., 2006; Daudi et al. ...
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Detection of ROS by DAB and NBT Staining in apples Leaves