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15 de abr. de 2014 · Introduction. Two dimensional polyacrylamide gel electrophoresis (2-DE) is considered a powerful tool used for separation and fractionation of complex protein mixtures from tissues, cells, or other biological samples. It allows separation of hundreds to thousands of proteins in one gel. This technique became more popular and comprehensive after ...
Gels with a higher acrylamide concentration (e.g. 20%) impede the movement of larger proteins to a greater degree than those of a smaller molecular weight but better resolve those of lower molecular weights (e.g. 4EBP1 ~ 20 kDa) (Chrambach & Rodbard 1971). Similarly, if the desired target is a large protein (e.g. mTOR ~289 KDa) a lower ...
Predicted molecular weight of the target protein needs to be taken into consideration for gel selection. Polyacrylamide gels can be considered as a filter with pore size that can be modulated by changing acrylamide percentage. Higher percentage will generate smaller pore size and will preferentially be used to separate protein with low ...
1 de abr. de 1970 · In this paper a technique is described for estimating the molecular weights of proteins by polyacrylamide gel electrophoresis. Electrophoresis was performed at pH 4.0 in 9 M urea at polyacrylamide concentrations ranging from 7 to 14% w v.Under these conditions it was found that the mobility of a protein varied exponentially as a function of the gel concentration.
The top portion of the gel is made of a stacking gel, made of a lower percentage of acrylamide. This allows the proteins to move faster and concentrate into a neat band prior to being separated in ...
It depends quite a bit on your protein, but as a starting point for 14kDa proteins I go with a 10% Bis-Tris gel in MES Buffer. As far as transferring, I use the classic wet transfer system.
High-molecular-weight proteins are better resolved with low-percentage acrylamide gels, whereas low-molecular-weight proteins are best resolved with a high percentage of acrylamide. Gradient gels are best for protein samples with a wide range of molecular weights.
