Western Blot Handbook
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Supplementary Materials and Method Immunostaining and Western Blot
Supplementary Materials and Method Immunostaining and Western Blot Analysis For immunofluorescence staining, mouse and human cells were fixed with 4% paraformaldehyde- PBS for 15 min. Following Triton-X100 permeabilization and blocking, cells were incubated with primary antibodies overnight at 4°C following with Alexa 594-conjugated secondary antibodies at 4°C for 1 hour (Thermo Fisher Scientific, 1:1000). Samples were mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) and immunofluorescence was detected using Olympus confocal microscopy. For western blot analysis, cells were lysed on ice using RIPA buffer supplemented with protease and phosphatase inhibitors (Sigma). Primary Antibodies for Immunostaining and Western Blot Analysis: Yap (14074, Cell Signaling), pYAP (4911, Cell Signaling), Lats1 (3477, Cell Signaling), pLats1( 8654, Cell Signaling), Wnt5a (2530, Cell Signaling), cleaved Caspase-3 (9661, Cell Signaling), Ki-67 (VP-K451, Vector Laboratories), Cyr61 (sc-13100, Santa Cruz Biotechnology), CTGF (sc-14939, Santa Cruz Biotechnology), AXL (8661, Cell Signaling), pErk (4376, Cell Signaling), pMEK (4376, Cell Signaling), Ck-19 (16858-1-AP, Proteintech), Actin (A2228, Sigma Aldrich), Vinculin (V4139, Sigma Aldrich), Kras (sc-30, Santa Cruz Biotechnology). Ectopic expression of YAP1 and WNT5A in mouse and human cells To generate YAP1S127A-expressing stable Pa04C cells, Pa04C cells were transfected with a linearized pcDNA3.1 plasmid with or without YAP1 cDNA containing S127A substitution. Two days post-transfection using Lipofectamine1000, cultures were selected in G418 (Sigma) and single clones were picked and expanded for further analysis. Overexpression of YAPS127A or WNT5A in human or mouse cells other than Pa04C were acheieved with lentivral infection. Briefly, lentivirus infection was performed by transfecting 293T cells with either GFP control, YAP1S127A, or WNT5A cloned in pHAGE lentivirus vector {EF1α promoter-GW-IRES-eGFP (GW: Gateway modified)}. -
Agarose Gel Electrophoresis
Laboratory for Environmental Pathogen Research Department of Environmental Sciences University of Toledo Agarose gel electrophoresis Background information Agarose gel electrophoresis of DNA is used to determine the presence and distinguish the type of nucleic acids obtained after extraction and to analyze restriction digestion products. Desired DNA fragments can be physically isolated for various purposes such as sequencing, probe preparation, or for cloning fragments into other vectors. Both agarose and polyacrylamide gels are used for DNA analysis. Agarose gels are usually run to size larger fragments (greater than 200 bp) and polyacrylamide gels are run to size fragments less than 200 bp. Typically agarose gels are used for most purposes and polyacrylamide gels are used when small fragments, such as digests of 16S rRNA genes, are being distinguished. There are also specialty agaroses made by FMC (e.g., Metaphor) for separating small fragments. Regular agarose gels may range in concentration from 0.6 to 3.0%. Pouring gels at less or greater than these percentages presents handling problems (e.g., 0.4% agarose for genomic DNA partial digests requires a layer of supporting 0.8% gel). For normal samples make agarose gels at 0.7%. The chart below illustrates the optimal concentrations for fragment size separation. The values listed are approximate and can vary depending on the reference that is used. If you do not know your fragment sizes then the best approach is to start with a 0.7% gel and change subsequently if the desired separation is not achieved. Nucleic acids must be stained prior to visualization. Most laboratories use ethidium bromide but other stains (e.g., SYBR green, GelStar) are available. -
SOP Template Northern Blotting with P-32
Standard Operating Procedure Procedure Northern Blotting using 32P Department Location SOP Prepared By: Section 1: Purpose Northern blotting is a standard method for the detection and quantification of RNA from a cell. This is done by isolating and purifying RNA and using a radioactively-labeled DNA or RNA probe to hybridize to and detect the RNA. Using this technique, temporal and spatial location of RNA expression can be found8. Unlike RT-PCR (real-time PCR), which quantifies the amount of RNA or DNA at various times, Northern blotting can be used not only to quantify but also determine the size of the RNA. It is also useful to perform Northern blotting when studying transfer RNA (tRNA), which appears as the two lowest bands on a gel1. The probes used in Northern blotting do not have to be radioactive, but radioactive probes still have the greatest sensitivity. Quantifying mRNA of tRNA can provide insight into gene expression in cells that are exposed to different environments. Section 2: Personal Protective Equipment and Survey Equipment PPE: • Nitrile Gloves • Lab coat or lab gown • Proper enclosed shoes • Safety glasses Other Equipment: • Geiger counter • Personal chest dosimeter Section 3: Radioactive Material 32P, specific type varies according to what procedure is followed for probe design Supplier: Perkin Elmer Starting activity: 10 mCi/mL Typical use quantities: no more than 10uCi per experiment • Radioactive material (RAM) benchtop exposure time: ~10-20 minutes Activity used per experiment: _______________ RAM handling time: _______________ Frequency of experiment: _______________ Section 4: Potential Hazards • 32P is a high-energy beta emitter and has a half-life of 14.29 days. -
Glycoprotein Detection with the Odyssey Infrared Imaging System
Glycoprotein Detection with the Odyssey ® Infrared Imaging System Julie A. Champoux, Kristi L.H. Ambroz, Joseph B. Hwang, William M. Volcheck, and Amy R. Schutz-Geshwender LI-COR Biosciences, Lincoln, NE 68504 INTRODUCTION bination of lectins can also be used to dissect the Glycosylation is one of the most common and carbohydrate composition of a target protein. important events in post-translational modifica- For more information about IRDye products tion, with over half of all proteins believed to be used in this study, go to http://www.licor.com/bio/ glycosylated.1 Cellular glycoconjugates play reagents/irdyes.jsp. important roles in many biological processes and have been implicated in cancer development, MATERIALS AND METHODS retrovirus infection and other diseases. Carbohy- Sensitivity of biotinylated Con A / IRDye drate-binding proteins known as lectins bind to 800CW-streptavidin and IRDye 800CW-Con A specific oligosaccharides, and can serve as markers Two-fold serial dilutions of α2-macroglobulin, to identify certain cell types or cellular compo- glucose oxidase and RNAse B (Sigma Cat# nents. Lectins have very high binding specificity M6159, 49178 and NEB Cat# P7817S were and have been used to characterize and purify mixed and separated on 4-12% polyacrylamide oligosaccharides.2 This application note describes Tris-glycine gels (Novex/Invitrogen). Samples the use of lectins to detect glycoproteins in West- were electrophoretically transferred onto nitro- ern blot format using the Odyssey® Infrared cellulose membrane (Osmonics). For all blots in Imaging System. this study, blocking was carried out with Odyssey Concanavalin A (Con A) is a carbohydrate- Blocking Buffer (LI-COR, Part # 927-40000) for 1 h binding protein that binds specifically to the at room temperature; washing was performed in most commonly occurring sugars: α-D-mannose, PBST (PBS + 0.1% Tween®-20) for 4 x 5 min, fol- α-D-glucose and, with lower affinity, α-N-acetyl- lowed by a 5 min rinse in PBS before imaging. -
Blotting Techniques Blotting Is the Technique in Which Nucleic Acids Or
Blotting techniques Blotting is the technique in which nucleic acids or proteins are immobilized onto a solid support generally nylon or nitrocellulose membranes. Blotting of nucleic acid is the central technique for hybridization studies. Nucleic acid labeling and hybridization on membranes have formed the basis for a range of experimental techniques involving understanding of gene expression, organization, etc. Identifying and measuring specific proteins in complex biological mixtures, such as blood, have long been important goals in scientific and diagnostic practice. More recently the identification of abnormal genes in genomic DNA has become increasingly important in clinical research and genetic counseling. Blotting techniques are used to identify unique proteins and nucleic acid sequences. They have been developed to be highly specific and sensitive and have become important tools in both molecular biology and clinical research. General principle The blotting methods are fairly simple and usually consist of four separate steps: electrophoretic separation of protein or of nucleic acid fragments in the sample; transfer to and immobilization on paper support; binding of analytical probe to target molecule on paper; and visualization of bound probe. Molecules in a sample are first separated by electrophoresis and then transferred on to an easily handled support medium or membrane. This immobilizes the protein or DNA fragments, provides a faithful replica of the original separation, and facilitates subsequent biochemical analysis. After being transferred to the support medium the immobilized protein or nucleic acid fragment is localized by the use of probes, such as antibodies or DNA, that specifically bind to the molecule of interest. Finally, the position of the probe that is bound to the immobilized target molecule is visualized usually by autoradiography. -
Microchip Electrophoresis
Entry Microchip Electrophoresis Sammer-ul Hassan Mechanical Engineering, University of Southampton, Southampton SO17 1BJ, UK; [email protected] Definition: Microchip electrophoresis (MCE) is a miniaturized form of capillary electrophoresis. Electrophoresis is a common technique to separate macromolecules such as nucleic acids (DNA, RNA) and proteins. This technique has become a routine method for DNA size fragmenting and separating protein mixtures in most laboratories around the world. The application of higher voltages in MCE achieves faster and efficient electrophoretic separations. Keywords: electrophoresis; microchip electrophoresis; microfluidics; microfabrications 1. Introduction Electrophoresis is an analytical technique that has been applied to resolve complex mixtures containing DNA, proteins, and other chemical or biological species. Since its discovery in the 1930s by Arne [1], traditional slab gel electrophoresis (SGE) has been widely used until today. Meanwhile, new separation techniques based on electrophoresis continue to be developed in the 21st century, especially in life sciences. Capillary electrophoresis (CE) provides a higher resolution of the separated analytes and allows the automation of the operation. Thus, it has been widely used to characterize proteins and peptides [2], biopharmaceutical drugs [3], nucleic acids [4], and the genome [5]. The development of microfabrication techniques has led to the further miniaturization of electrophoresis known Citation: Hassan, S.-u. Microchip as microchip electrophoresis (MCE). MCE offers many advantages over conventional Electrophoresis. Encyclopedia 2021, 1, capillary electrophoresis techniques such as the integration of different separation functions 30–41. https://dx.doi.org/10.3390/ onto the chip, the consumption of small amounts of sample and reagents, faster analyses encyclopedia1010006 and efficient separations [6,7]. -
Near-Infrared Fluorescent Northern Blot
Downloaded from rnajournal.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press METHOD Near-infrared fluorescent northern blot BRET R. MILLER,1,4 TIANQI WEI,1,4 CHRISTOPHER J. FIELDS,1,2 PEIKE SHENG,1,2 and MINGYI XIE1,2,3 1Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA 2UF Health Cancer Center, University of Florida, Gainesville, Florida 32610, USA 3UF Genetics Institute, University of Florida, Gainesville, Florida 32610, USA ABSTRACT Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive 32P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than 32P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present al- ternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of dif- ferent RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis. -
NORTHERN BLOT SEM.I Dr.Ramesh Pathak the Northern Blot Is Also
NORTHERN BLOT SEM.I Dr.Ramesh Pathak The Northern blot is also called RNA blot. It is a technique used in molecular biology to study gene expression by detection of RNA in a sample. The northern blot technique developed by James Alwine, David Kemp and Jeorge Stark at Stanford University with contribution from Gerhard Heinrich in 1977.Northern blotting takes its name from its similarity to the first blotting technique, the southern blotting named after biologist Edwin Southern. Procedure The blotting process starts with extraction of total RNA from a homogenized tissue sample or cells on agarose gel containing formaldehyde which is a denaturing agent for the RNA to limit secondary structure. Eukaryotic mRNA can then be isolated through the use of oligo(dT) cellulose chromatography to isolate only those RNAs with a poly A tail. RNA samples are then separated by gel electrophoresis. Since the gels are fragile and the probes are unable to enter matrix, the RNA samples, now separated by size, are transferred to nylon membrane through a capillary or vacuum blotting system. A nylon membrane with a positive charge, is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them. The transfer buffer used for the blotting usually contain formamide because it lowers the annealing temperature of the probe-RNA interaction, thus eliminating the need for high temperature, which could cause RNA degradation. Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat. -
Western Blotting Guidebook
Western Blotting Guidebook Substrate Substrate Secondary Secondary Antibody Antibody Primary Primary Antibody Antibody Protein A Protein B 1 About Azure Biosystems At Azure Biosystems, we develop easy-to-use, high-performance imaging systems and high-quality reagents for life science research. By bringing a fresh approach to instrument design, technology, and user interface, we move past incremental improvements and go straight to innovations that substantially advance what a scientist can do. And in focusing on getting the highest quality data from these instruments—low backgrounds, sensitive detection, robust quantitation—we’ve created a line of reagents that consistently delivers reproducible results and streamlines workflows. Providing scientists around the globe with high-caliber products for life science research, Azure Biosystems’ innovations open the door to boundless scientific insights. Learn more at azurebiosystems.com. cSeries Imagers Sapphire Ao Absorbance Reagents & Biomolecular Imager Microplate Reader Blotting Accessories Corporate Headquarters 6747 Sierra Court Phone: (925) 307-7127 Please send purchase orders to: Suite A-B (9am–4pm Pacific time) [email protected] Dublin, CA 94568 To dial from outside of the US: For product inquiries, please email USA +1 925 307 7127 [email protected] FAX: (925) 905-1816 www.azurebiosystems.com • [email protected] Copyright © 2018 Azure Biosystems. All rights reserved. The Azure Biosystems logo, Azure Biosystems™, cSeries™, Sapphire™ and Radiance™ are trademarks of Azure Biosystems, Inc. More information about Azure Biosystems intellectual property assets, including patents, trademarks and copyrights, is available at www.azurebiosystems.com or by contacting us by phone or email. All other trademarks are property of their respective owners. -
QPNC-PAGE Standardized Protocol for Acidic, Basic and Neutral Metalloproteins MW 6 - > 200Kda USE ONLY FRESH GELS
QPNC-PAGE Standardized Protocol For Acidic, Basic and Neutral Metalloproteins MW 6 - > 200kDa USE ONLY FRESH GELS Correspondence: Bernd Kastenholz • Research Centre Juelich • Institute of Chemistry and Dynamics of the Geosphere – Phytosphere (ICG-3) • Juelich 52425 • Germany • E-mail: [email protected] Recommended Tools (Bio-Rad) Model 491 Prep Cell (U.S. patent no. 4,877,510) Power Pac 1000: Constant Power: 5 W; Time: 8 hr Model EP-1 Econo Pump: 1 mL/min; 5 mL/fraction; 80 mL prerun V; 480 ml total V (Eluent) Model 2110 Fraction Collector: 80 Fractions Model EM-1 Econo UV Monitor: AUFS 1.0; Detection Wavelength 254 nm Model 1327 Econo Recorder: Range: 100 mV; Chart Speed: 6 cm/hr Model SV-3 Diverter Ventil Buffer Recirculation Pump: Flow Rate: 95 mL/min (Electrophoresis Buffer) Stock Solutions 1) 200 mM Tris-HCl 10 mM NaN3 pH 10.00 – Keep RT. 2) 200 mM Tris-HCl 10 mM NaN3 pH 8.00 – Keep RT. 3) 40 % Acrylamide/Bis 2.67 % C - Keep 4°C. 4) 10 % Ammonium Persulfate (APS) - Keep 4°C (freshly prepared). Electrophoresis Buffer 20 mM Tris-HCL 1 mM NaN3 pH 10.00 degassed – Keep 4°C Upper Electrophoresis Chamber (Prep Cell): 500 mL Electrophoresis Buffer Lower Electrophoresis Chamber (Prep Cell): 2000 mL Electrophoresis Buffer Eluent 20 mM Tris-HCL 1 mM NaN3 pH 8.00 degassed – Keep 4°C Elution Chamber (Prep Cell): 700 mL Elution Buffer Separating Gel Acrylamide 4% T Volume: 40 mL ingredients: 4 mL 40 % Acrylamide/Bis 2.67 % C 4 mL 200 mM Tris-HCl 10 mM NaN3 pH 10.00 32 mL H2O 200 µL 10% APS 20 µL TEMED Add TEMED and APS at the end. -
High Sensitivity Protein Gel Electrophoresis Label Compatible with Mass-Spectrometry
biosensors Brief Report High Sensitivity Protein Gel Electrophoresis Label Compatible with Mass-Spectrometry Joshua A. Welsh 1 , Lisa M. Jenkins 2 , Julia Kepley 1, Gaelyn C. Lyons 2, David M. Moore 1 , Tim Traynor 1, Jay A. Berzofsky 3 and Jennifer C. Jones 1,* 1 Laboratory of Pathology, Centre for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] (J.A.W.); [email protected] (J.K.); [email protected] (D.M.M.); [email protected] (T.T.) 2 Laboratory of Cell Biology, Centre for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] (L.M.J.); [email protected] (G.C.L.) 3 Vaccine Branch, Centre for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] * Correspondence: [email protected] Received: 20 August 2020; Accepted: 28 October 2020; Published: 31 October 2020 Abstract: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely utilized technique for macromolecule and protein analysis. While multiple methods exist to visualize the separated protein bands on gels, one of most popular methods of staining the proteins is with Coomassie dye. A more recent approach is to use Bio-Rad stain-free technology for visualizing protein bands with UV light and achieve similar or greater sensitivity than that of Coomassie dye. Here, we developed a method to further amplify the sensitivity of stain-free gels using carboxyfluorescein succinimidyl ester (CFSE) staining. We compared our novel method using foetal bovine serum samples with Coomassie dye, standard stain-free gels, and silver staining. -
Gene Expression Detection Methods RNA
Gene expression detection methods RNA Dr. Ingrid Müller - AG von Laer 1 TRANSCRIPTIONAL CONTROL REGULATES DIFFERENTIATION Four different human cells - same genes, different structures and functions due to differential gene expression 2 THE CENTRAL DOGMA • Flow of Information: DNA Replication Transcription Translation A B Cells in all living organisms are continually activating or deactivating genes through gene expression, which contain the information required for producing proteins through proteins synthesis. When a particular protein is required by the cell, the gene coding for that protein is activated. The first stage in producing a protein involves the production of an RNA copy of the gene's DNA sequence. This RNA copy is the messenger RNA. The amount of mRNA produced correlates with the amount of protein eventually synthesised and measuring the amount of a particular mRNA produced by a given cell or tissue is often easier than measuring the amount of the final protein. Levels in gene activation may vary between cancerogenic and healthy cells. 3 MOLECULAR METHODS TOOL BOX I. Analysis II. Overexpression III. Inhibition •Western Blot •(adding proteins to in •pharmacological inhibitors, •Proteomics vitro reactions) •dominant-negative Protein •immuno- proteins, histochemisty • protein depletion using •protein chimera antibodies •Electrophoresis •microinjection •RNAi & •Northern Blot •siRNA •RNAse protection •Morpholinos RNA assays •Microarrays •RT-PCR •RNA in-situ •Reporter genes •(microinjection) •Knock-out •Electroporation, •Integrational mutagenesis DNA (gene) •lipofection, •Classic genetics •transgenics 4 RNA METHODS: ELECTROPHORESIS 5 PURIFICATION OF MESSENGER RNA USING OLIGO DT COLUMNS Total cellular RNA; apply at room temperature to Break open anneal polyA tail to oligo(dT) cells in the presence of RNAse Oligo(dT) inhibitors attached to AAAA.