A Guide to Polyacrylamide Gel Electrophoresis and Detection
Total Page:16
File Type:pdf, Size:1020Kb
Load more
Recommended publications
-
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. -
Validation of Rapid Enzymatic Quantification of Acetic Acid In
foods Article Validation of Rapid Enzymatic Quantification of Acetic Acid in Vinegar on Automated Spectrophotometric System Irene Dini 1,* , Ritamaria Di Lorenzo 1, Antonello Senatore 2, Daniele Coppola 2 and Sonia Laneri 1 1 Department of Pharmacy, University of Naples Federico II, Via Domenico Montesano 49, 80131 Napoli, Italy; [email protected] (R.D.L.); [email protected] (S.L.) 2 Lcm Laboratory for Product Analysis of Chamber of Commerce, Corso Meridionale, 80143 Napoli, Italy; [email protected] (A.S.); [email protected] (D.C.) * Correspondence: [email protected] Received: 9 May 2020; Accepted: 4 June 2020; Published: 9 June 2020 Abstract: Vinegar is produced from the fermentation of agricultural materials and diluted acetic acid (diluted with water to 4–30% by volume) via sequential ethanol and acetic acid fermentation. The concentration of acetic acid must be measured during vinegar production. A Community method for analyzing acetic acid in vinegar is a non-specific method based on the assumption that the total acid concentration of the vinegar is attributable to the acetic acid. It consists of titration with a strong base in the presence of an indicator. This test is laborious and has a time-consuming character. In this work, a highly specific automated enzymatic method was validated, for the first time, to quantify the acetic acid in the wine vinegar, in terms of linearity, precision, repeatability, and uncertainty measurement. The results were compared to the Community method of analysis. Regression coefficient 1 and the normal distribution of residuals in the ANOVA test confirmed the method’s linearity. -
Gel-Syn™ Product Information Caution
GEL-SYN™ PRODUCT INFORMATION CAUTION: Federal law restricts this device to sale by or on the order of a physician (or properly licensed practitioner). CONTENT Each 1 mL of Gel-Syn contains: Sodium Hyaluronate: 8.4 mg Sodium Chloride: 8.5 mg Sodium Phosphate, Dibasic: 0.16 mg Sodium Phosphate, Monobasic: 0.045 mg Water for Injection: q.s. to 1.0 mL DESCRIPTION Gel-Syn is a sterile, buffered solution of highly purified sodium hyaluronate with a molecular weight of approximately 1100 kDa, obtained through fermentation of Streptococci of Lancefield groups A and C and chemically unmodified. INDICATION Gel-Syn is indicated for the treatment of pain in osteoarthritis (OA) of the knee in patients who have failed to respond adequately to conservative non-pharmacologic therapy and simple analgesics (e.g., acetaminophen). CONTRAINDICATIONS • Do not administer to patients with known hypersensitivity (allergy) to sodium hyaluronate preparations. • Do not inject Gel-Syn into the knees of patients having knee joint infections or skin diseases or infections in the area of the injection site. WARNINGS • Do not concomitantly use disinfectants containing quaternary ammonium salts for skin preparation because sodium hyaluronate can precipitate in their presence. • Inject into the synovial space only. Do not inject by intravascular route. • Do not inject outside the synovial space or into the synovial tissue or capsule. An extra- articular injection of the product can cause local adverse events. PRECAUTIONS General • The safety and effectiveness of Gel-Syn in locations other than the knee, and for conditions other than osteoarthritis, have not been established. • Strict aseptic administration technique must be followed. -
Caenorhabditis Elegans BRICHOS Domain–Containing Protein C09F5.1 Maintains Thermotolerance and Decreases Cytotoxicity of A42 B
G C A T T A C G G C A T genes Article Caenorhabditis elegans BRICHOS Domain–Containing Protein C09F5.1 Maintains Thermotolerance and Decreases Cytotoxicity of Aβ42 by Activating the UPR Myungchul Song 1, Kyunghee Song 1,2, Sunghee Kim 1,3, Jinyoung Lee 1,4, Sueyun Hwang 5 and Chingtack Han 1,* 1 Department of Life Science, Sogang University, Seoul 04107, Korea; [email protected] (M.S.); [email protected] (K.S.); [email protected] (S.K.); jinylee@amorepacific.com (J.L.) 2 LG Household & Health Care, Daejeon 34114, Korea 3 Department of Medicine, Biomedical Research Institute, Seoul National University Hospital, Seoul 03080, Korea 4 Amorepacific R&D Center, Yongin 17074, Korea 5 Department of Chemical Engineering, Hankyung National University, Anseong 17579, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-2-705-8454 Received: 11 December 2017; Accepted: 9 March 2018; Published: 13 March 2018 Abstract: Caenorhabditis elegans C09F5.1 is a nematode-specific gene that encodes a type II transmembrane protein containing the BRICHOS domain. The gene was isolated as a heat-sensitive mutant, but the function of the protein remained unclear. We examined the expression pattern and subcellular localization of C09F5.1 as well as its roles in thermotolerance and chaperone function. Expression of C09F5.1 under heat shock conditions was induced in a heat shock factor 1 (HSF-1)–dependent manner. However, under normal growth conditions, most cells types exposed to mechanical stimuli expressed C09F5.1. Knockdown of C09F5.1 expression or deletion of the N-terminal domain decreased thermotolerance. -
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. -
Clinical Pharmacology 1: Phase 1 Studies and Early Drug Development
Clinical Pharmacology 1: Phase 1 Studies and Early Drug Development Gerlie Gieser, Ph.D. Office of Clinical Pharmacology, Div. IV Objectives • Outline the Phase 1 studies conducted to characterize the Clinical Pharmacology of a drug; describe important design elements of and the information gained from these studies. • List the Clinical Pharmacology characteristics of an Ideal Drug • Describe how the Clinical Pharmacology information from Phase 1 can help design Phase 2/3 trials • Discuss the timing of Clinical Pharmacology studies during drug development, and provide examples of how the information generated could impact the overall clinical development plan and product labeling. Phase 1 of Drug Development CLINICAL DEVELOPMENT RESEARCH PRE POST AND CLINICAL APPROVAL 1 DISCOVERY DEVELOPMENT 2 3 PHASE e e e s s s a a a h h h P P P Clinical Pharmacology Studies Initial IND (first in human) NDA/BLA SUBMISSION Phase 1 – studies designed mainly to investigate the safety/tolerability (if possible, identify MTD), pharmacokinetics and pharmacodynamics of an investigational drug in humans Clinical Pharmacology • Study of the Pharmacokinetics (PK) and Pharmacodynamics (PD) of the drug in humans – PK: what the body does to the drug (Absorption, Distribution, Metabolism, Excretion) – PD: what the drug does to the body • PK and PD profiles of the drug are influenced by physicochemical properties of the drug, product/formulation, administration route, patient’s intrinsic and extrinsic factors (e.g., organ dysfunction, diseases, concomitant medications, -
Analysis of Proteins by Immunoprecipitation
Laboratory Procedures, PJ Hansen Laboratory - University of Florida Analysis of Proteins by Immunoprecipitation P.J. Hansen1 1Dept. of Animal Sciences, University of Florida Introduction Immunoprecipitation is a procedure by which peptides or proteins that react specifically with an antibody are removed from solution and examined for quantity or physical characteristics (molecular weight, isoelectric point, etc.). As usually practiced, the name of the procedure is a misnomer since removal of the antigen from solution does not depend upon the formation of an insoluble antibody-antigen complex. Rather, antibody-antigen complexes are removed from solution by addition of an insoluble form of an antibody binding protein such as Protein A, Protein G or second antibody (Figure 1). Thus, unlike other techniques based on immunoprecipitation, it is not necessary to determine the optimal antibody dilution that favors spontaneously-occurring immunoprecipitates. Figure 1. Schematic representation of the principle of immunoprecipitation. An antibody added to a mixture of radiolabeled (*) and unlabeled proteins binds specifically to its antigen (A) (left tube). Antibody- antigen complex is absorbed from solution through the addition of an immobilized antibody binding protein such as Protein A-Sepharose beads (middle panel). Upon centrifugation, the antibody-antigen complex is brought down in the pellet (right panel). Subsequent liberation of the antigen can be achieved by boiling the sample in the presence of SDS. Typically, the antigen is made radioactive before the immunoprecipitation procedure, either by culturing cells with radioactive precursor or by labeling the molecule after synthesis has been completed (e.g., by radioiodination to iodinate tyrosine residues or by sodium [3H]borohydride reduction to label carbohydrate). -
Physical-Chemical Characteristics of Whitening Toothpaste and Evaluation of Its Effects on Enamel Roughness
Dental materials Physical-chemical characteristics of whitening toothpaste and evaluation of its effects on enamel roughness Sérgio Paulo Hilgenberg(a) Abstract: This in vitro study evaluated the physical-chemical characteris- (a) Shelon Cristina Souza Pinto tics of whitening toothpastes and their effect on bovine enamel after ap- Paulo Vitor Farago(b) Fábio André Santos(a) plication of a bleaching agent (16% carbamide peroxide). Physical-chem- Denise Stadler Wambier(a) ical analysis was made considering mass loss by desiccation, ash content and pH of the toothpastes. Thirty bovine dental enamel fragments were prepared for roughness measurements. The samples were subjected to (a) Department of Dentistry, School of Dentistry, Ponta Grossa State University, bleaching treatments and simulated brushing: G1. Sorriso Dentes Brancos Ponta Grossa, PR, Brazil. (Conventional toothpaste), G2. Close-UP Whitening (Whitening tooth- (b) Department of Pharmacy, School of paste), and G3. Sensodyne Branqueador (Whitening toothpaste). The av- Dentistry, Ponta Grossa State University, erage roughness (Ra) was evaluated prior to the bleaching treatment and Ponta Grossa, PR, Brazil. after brushing. The results revealed differences in the physical-chemical characteristics of the toothpastes (p < 0.0001). The final Ra had higher values (p < 0.05) following the procedures. The mean of the Ra did not show significant differences, considering toothpaste groups and bleach- ing treatment. Interaction (toothpaste and bleaching treatment) showed significant difference -
Optical Properties and Denaturation by Guanidinium Chloride and Urea
Biochem. J. (1977) 161, 321-331 321 Printed in Great Britain Optical Properties and Denaturation by Guanidinium Chloride and Urea of the Adenosine Triphosphatase of Micrococcus lysodeikticus A COMPARISON OF FOUR MOLECULAR FORMS OF THE ENZYME By MANUEL NIETO and JUAN A. AYALA Seccion de Bioquimica de Membranas, Centro de Investigaciones Biol6gicas, Veldzquez 144, Madrid-6, Spain (Received 7 July 1976) 1. The fluorescence and circular dichroism offour homogeneous preparations of ATPase (adenosine triphosphatase) fromMicrococcus lysodeikticus differing in molecular structure and enzymic properties were examined at pH 7.5 and 25°C. Emission was maximum at 325 and 335nm and the relative intensities at these wavelengths may be used to characterize the different ATPase preparations. The circular-dichroism spectra exhibited negative extrema at 208 and 220nm, and the relative value of the molar ellipticity at these wave- lengths was also different for each molecular form ofthe enzyme. 2. The four preparations undergo two consecutive major unfolding transitions in guanidinium chloride (midpoints at 0.94 and 1.5 M denaturant), with concomitant destruction ofthe quaternary structure of the protein. A comparatively minor alteration in the ATPase structure also occurred in 0.05-0.2M-guanidine and led to complete inactivation ofthe enzyme. The inactivation and the first unfolding transition were reversible by dilution of the denaturant; the transition with midpoint at 1.5M-guanidine was irreversible. 3. Similar results were obtained in urea, except that the successive transitions had midpoints at concentrations of denaturant of 0.4, 2.0 and 4.5M. Low concentrations of urea caused a noticeable activation of the enzyme activity and alterations of the electrophoretic mobility of the ATPase. -
Modified Bradford Assay Method of Protein Quantification Utilising Dye Reagents from Four Nigerian Plants
International Journal of Research Studies in Biosciences (IJRSB) Volume 3, Issue 12, December 2015, PP 79-87 ISSN 2349-0357 (Print) & ISSN 2349-0365 (Online) www.arcjournals.org Modified Bradford Assay Method of Protein Quantification Utilising Dye Reagents from Four Nigerian Plants *S. O. Okeniyi+, *J. Ogbodobri, **A. O. Oyedeji, *P. E. Omale, *M. M. Adeyemi, *S. Garba, ***J. A. Lori *Department.of Chemistry, Nigerian Defence Academy, Afaka, Kaduna State - Nigeria ** Department of Chemical & Physical Sciences, Walter Sisulu University Eastern Cape, South Africa ***Department of Chemistry, Bingham University, Karu, Nassarawa State - Nigeria Abstract: Aqueous and organic solvents extraction process using ethanol, methanol and chloroform were carried out with four different Nigerian plants namely: Pterocarpus osun (uhe), Lawsonia inermis (lalle), Bixa Orellana (annatto) and Hibiscus sabderriffa (zobo) to extract dye reagents from the plants. The ability of the dye reagents to replace Coomassie Brilliant Blue in the Bradford assay method of protein quantification were determined and compared. The solvents extracts gave good colourful results in the extraction of the dye reagents while only aqueous extract of Hisbiscus sabderiffa (zobo) gave similar results to that of solvent extracts. The solvent extracts obtained from Pterocarpus osun (uhe), Lawsonia inermis (lalle) and Bixa Orellana (annatto) plants could not be used to estimate amino acids from protein samples. However, solvent extracts of Hibiscus sabderriffa (zobo) was able to estimate amino acids from protein samples. The change in maximum wavelength (λmax) and the increased absorption with zobo dye reagent; on addition of protein samples showed that solvent extract of Hibiscus sabderiffa (zobo) dye has the potential to quantify and estimate amino acids in protein samples as much as the Coomassie Blue utilised in the Bradford assay method. -
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. -
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].