Gel Electrophoresis of Protein - From Basic Science to Practical Approach

Kavoosi, Gholamreza; Ardestani, Sussan Kaboudanian

doi:10.5772/38062

Cited by 4 publications

(2 citation statements)

References 33 publications

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“…The resolving gel of pH 8.8 was first prepared and allowed for polymerization. Similarly the stacking gel pH 6.8 was allowed to polymerize on the top of first gel using 7.5 % acryl amide and bis-acryl amide concentration as described in the literature (Alhashem et al, 2022;Davis, 1964;Kavoosi & Ardestani, 2012). Ion exchange chromatography: Out of five, one pooled fraction (fraction no.5) was not homogenized and this was separated by CM-Sephadex C-50 column (26 x 2cm), which was previously equilibrated with sodium phosphate buffer pH 7.0.…”

Section: Determination Of Proteinmentioning

confidence: 99%

Purification and Characterization of Alkaline Protease Isolated from Cotton (Gossypium hirsutum) Seeds

Shaikh,

Dahot,

Sajid

et al. 2023

joarps

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Proteases are widely utilized both in physiological and commercial fields such as medicine, food, detergent, and leather. Plant-originated proteases play a significant role in several biomedical fields due to their easy accessibility and activity. Pakistan is an agro-based country and can be an ideal place for the isolation of industrially important proteases from plant sources such as cotton, which is the main crop and frequently available and low cost. Purification of protease was carried out by fractionation with two-fold acetone, ethanol, methanol and various concentrations (40-80%) of ammonium sulphate. The precipitates formed were collected after centrifugation and dialyzed for 24 hours against universal buffer pH 7.0 and was centrifuged in a cooled refrigerated. The dialyzed sample was loaded on Sephadex G–100 gel column. The fractions of the samples were collected and their absorbance of protein was monitored at 280 nm. The homogeneity of the purified enzyme was checked by SDS gel electrophoresis The purified protease enzyme has optimum activity at 30°C and pH 8.0 when casein was used as substrate. The Km and Vmax values of purified cotton seed's alkaline protease activity was recorded as 0.03M and 17 μmol/minute respectively. Protease activity was increased by the addition of cysteine but inhibited by Iodoacetic acid and β-Mercaptoethanol and decreased with some metal ions. These characteristics of the purified enzyme allowed classifying it as a cysteine protease. In conclusion, this study suggests that the alkaline protease enzyme is the best choice for commercial use

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Section: Determination Of Proteinmentioning

confidence: 99%

Purification and Characterization of Alkaline Protease Isolated from Cotton (Gossypium hirsutum) Seeds

Shaikh,

Dahot,

Sajid

et al. 2023

joarps

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show abstract

“…La polimerización se inicia por la liberación de radicales libres a partir del persulfato de amonio (PSA) por la acción del catalizador N,N,N',N'tetrametiletilendiamina (TEMED) (FIGURA 14).El tamaño de poro del gel y por tanto su rango de separación viene determinada por la proporción de acrilamida + bisacrilamida (T %), siendo menor el poro cuanto mayor sea este porcentaje. La mayoría de proteínas se separan bien en un rango del 5-12%[181].FIGURA 14. Reacción de polimerización de la acrilamidaLa detección de proteínas y glicoproteínas se realiza mayoritariamente mediante electroforesis en gel de poliacrilamida en condiciones desnaturalizantes en presencia de sodio dodecil sulfato o SDS (SDS-PAGE), donde la separación de las biomoléculas viene determinada exclusivamente por su masa molecular y por el tamaño de poro del gel[182].El SDS es un detergente aniónico fuerte que desnaturaliza las proteínas y rompe las interacciones no-movilidad de la glicoproteína durante la electroforesis que se comportará como si tuviera un peso molecular mayor, pudiéndose sobrestimar su tamaño hasta en un 30%. Las cadenas laterales de los carbohidratos pueden interaccionar de forma inespecífica con componentes del gel de electroforesis, perturbando su migración durante la electroforesis, dando lugar a la dispersión de las bandas que resultan ser más anchas de…”

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Extracción y caracterización de polisacáridos y estudio del perfil de compuestos volátiles en hongos comestibles

Romero¹

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Electrophoresis and Blotting of DNA

Sedivy-Haley

Tamber

Hancock

2013

Encyclopedia of Life Sciences

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Deoxyribonucleic acid (DNA) electrophoresis and blotting are techniques commonly used to visualise DNA. Both techniques use simple, inexpensive protocols that rely on fundamental properties of DNA, and these protocols have changed little since they were introduced. Electrophoresis relies on the negative electrical charge of DNA to draw these molecules through a gel, separating DNA molecules on the basis of size. Southern blotting makes use of sequence complementarity to identify specific DNA fragments on the basis of their sequences. Despite the introduction of more powerful methods such as polymerase chain reaction and DNA sequencing, electrophoresis and blotting techniques are still widely used to identify DNA fragments of interest, including the identification of unusual structures within DNA and the analysis of larger fragments which are not easily analysed by other methods. Key Concepts: DNA electrophoresis and blotting are fundamental methods of molecular biology. Basic physical and chemical properties of DNA are used to analyse unknown sequences. Gel electrophoresis uses an electrical current to separate DNA by size. Southern blotting locates a specific DNA sequence on a gel using a probe sequence that is its chemical match. Minor variations on these procedures can improve results for specific experiments. These techniques are still used to analyse unknown DNA molecules and identify DNA fragments of interest. The principles behind electrophoresis and blotting can also be seen in newer methods.

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Gel Electrophoresis of Protein - From Basic Science to Practical Approach

Cited by 4 publications

References 33 publications

Purification and Characterization of Alkaline Protease Isolated from Cotton (Gossypium hirsutum) Seeds

Purification and Characterization of Alkaline Protease Isolated from Cotton (Gossypium hirsutum) Seeds

Extracción y caracterización de polisacáridos y estudio del perfil de compuestos volátiles en hongos comestibles

Electrophoresis and Blotting of DNA

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