POLYACRYLAMIDE GELS

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Automatic analysis of 2D polyacrylamide gels in the diagnosis of DNA polymorphisms

Automatic analysis of 2D polyacrylamide gels in the diagnosis of DNA polymorphisms

Modern DNA analysis is used in many areas of life sciences, from biology [1] to foren- sic medicine or microwave analysis [2,3]. For many such cases, the analysis of DNA is associated with electrophoresis carried out on polyacrylamide gels, an universal analyt- ical technique used to separate DNA fragments by size. The advantages of using poly- acrylamide gels are low cost of staining separated DNA fractions and also easily interpretable analysis results. The obtained results are compared manually or semi- automatically. The manual method involves a manual selection of lanes and bands that are in the analysed area. Most often this occurs by selecting interesting bands on the printed analysis result (Figure 1) or by placing the lines along the lane on the computer

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Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

To electrophoretically fractionate small DNA molecules such as those obtained by polymerase chain reaction (PCR), polyacrylamide gels offer the highest resolution power. As polyacrylamide pore size is indipendent on the buffer used [6], electrophoretic fractionations were per- formed indifferently in TAE or TBE buffer. As the use of SDS-PAGE in DNA electrophoretic fractionation was sug- gested to enhance electrophoretic resolution power [7], we prepared horizontal polyacrylamide gels both in the presence or absence of SDS. As shown in Figure 4, DNA electrophoretic fractionation using 6 cm long gels in our apparatus, separates molecules which differ by only a few nucleotides.

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Rapid Electrophoretic Staining and Destaining of Polyacrylamide Gels

Rapid Electrophoretic Staining and Destaining of Polyacrylamide Gels

Polyacrylamide gel electrophoresis (PAGE) is one of the most widely used methods to analyze proteins biochemically [1]. After separation by electrophoresis, protein bands need to be detected with a subsequent staining step. A number of staining methods for PAGE gels have been reported, such as Coomassie brilliant blue (CBB), amido black, and silver staining [2–4]. Coomassie brilliant blue staining has been commonly used to stain protein bands in PAGE gels, due to its low cost, easy operation, and relatively high sensitivity [5]. However, according to the typical protocol, the staining and destaining steps require a long time (from 4 h to overnight). Several rapid staining and destaining methods with reduced processing times have been reported. Hervieu reported that CBB-stained sodium dodecyl sulfate (SDS)-PAGE gels could be destained by boiling in distilled water [6]. It has been reported that heat treatment accelerates the staining and destaining steps [7,8]. A modified, rapid Fairbanks Coomassie stain, using a microwave oven, gave a sensitivity limit of 5 ng [9,10]. However, boiling a solution containing methanol is hazardous to one’s health. Dong et al. reported CBB-staining and destaining at boiling temperature without acid and methanol [11]. Lawrence et al. reported a staining method using CBB G-250 with a low concentration of hydrochloric acid, instead of acetic acid [12]. In these procedures, the hot destaining solution should be exchanged repeatedly, leading to risk of burn injury. Kang et al. reported a sensitive colloidal staining method using CBB G-250, using less-toxic ethanol instead of methanol [13,14]. Although this staining method is very sensitive, up to ~1 ng, more than two hours of incubation is required for 80% staining. Several companies produce rapid protein staining/destaining systems, such as eStain™ from GenScript (Piscataway, NJ, USA, Cat. no.: L00657) or Pierce™ Power Stainer from Thermo Scientific (Waltham, MA, USA, Cat. no.: 22840). However, the electrophoretic apparatuses are expensive (~$2000), and each staining costs ~$5 per gel.

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Biochemical and Immunological Properties of Human Terminal Deoxynucleotidyl Transferase Purified from Blasts of Acute Lymphoblastic and Chronic Myelogenous Leukemia

Biochemical and Immunological Properties of Human Terminal Deoxynucleotidyl Transferase Purified from Blasts of Acute Lymphoblastic and Chronic Myelogenous Leukemia

Terminal deoxynucleotidyl transferase was purified to homogeneity from the blasts of eight patients with leukemia and compared with purified transferase from normal human and calf thymus. In two cases phenylmethanesulfonylfluoride was added during purification to reduce proteolysis. Comparative kinetic analyses of the purified enzymes indicated no differences in catalytic properties. There was substantial variation in the molecular structure of terminal transferase on denaturing polyacrylamide gels: (a) a protein that migrated as a single polypeptide with M r = 62,000 was isolated from two patients with acute lymphoblastic leukemia and from MOLT-4 cells; (b) a protein that migrated as a single polypeptide with M r = 42,500 was isolated from two patients with acute lymphoblastic leukemia; (c) a protein that migrated as a single polypeptide with M r = 42,500 was isolated from two patients with

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Studies of Water Holding in Polyacrylamide and Heat-Induced Protein Gels.

Studies of Water Holding in Polyacrylamide and Heat-Induced Protein Gels.

The ability of food gels to hold water affects product yield and organoleptic quality. Most workers believe water is held by capillarity, such that gels having smaller mean pore diameter and a more hydrophilic surface hold water more tightly. To date however only qualitative evidence relating pore size to water holding (WH) properties has been provided. We sought to provide quantitative confirmation of this hypothesis, using scanning electron microscopy coupled with image analysis to measure pore size, and measuring contact angles by the captive bubble method, in both model polyacrylamide gels and heat-induced protein (minced chicken breast) gels. WH was assessed as water lost during cooking (cook loss, CL, of meat gels) or upon centrifugation (expressible water, EW) or capillary suction (CSL) for all prepared gels. As predicted by the capillarity hypothesis, gels with lower water losses exhibited a more hydrophilic surface (smaller contact angle). Yet greater CL and EW correlated with smaller, not larger mean pore diameter of gels. The capillary suction method proved more sensitive to measuring small differences in WH of prepared gels.

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GENE TYPING   TWO DIMENSIONAL ELECTROPHORESIS pdf

GENE TYPING TWO DIMENSIONAL ELECTROPHORESIS pdf

Recently, various systems have been proposed to analyse gene-coding and regulatory sequences more effectively for all possible variations. Here we review the development and application of one such system, two-dimensional gene scanning (TDGS). This method is based on the two-dimensional separation of polymerase chain reaction (PCR)-ampli R ed gene fragments on the basis of both size and base pair sequence in polyacrylamide gels. Attention will be focused on most recent developments in automation and miniaturization of the two-dimensional elec- trophoresis procedure. Future developments towards a dedicated fully automated high-throughput system for gene analysis will be discussed.

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Agarose Gels pdf

Agarose Gels pdf

two decades. Improved imaging systems and labelling agents for high sensitivity detection and linear quantitation by S uorescence and chemiluminescence offer attractive alternatives to immunostaining and the use of radioisotopes, and have made rapid auto- mated nucleic acid sequencing possible. Except for those involved in nucleic acid work, few laboratories perform electrophoresis on agarose on a day-to-day basis; thus, accessories for many techniques such as immunoelectrophoresis have been dropped by sup- pliers. There may be a revival because of growing interest in several proteins which are not separable in polyacrylamide matrices, von Willebrand factor multimers for diagnosis of certain types of von Willebrand’s disease, and high molecular mass derivatives of R brinogen as markers of vascular disease.

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