About: Isoelectric point is a research topic. Over the lifetime, 12854 publications have been published within this topic receiving 449870 citations.
Papers published on a yearly basis
TL;DR: This technique provides a method for estimation of the number of proteins made by any biological system and can resolve proteins differing in a single charge and consequently can be used in the analysis of in vivo modifications resulting in a change in charge.
Abstract: A technique has been developed for the separation of proteins by two-dimensional polyacrylamide gel electrophoresis. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources. Proteins are separated according to isoelectric point by isoelectric focusing in the first dimension, and according to molecular weight by sodium dodecyl sulfate electrophoresis in the second dimension. Since these two parameters are unrelated, it is possible to obtain an almost uniform distribution of protein spots across a two-diminsional gel. This technique has resolved 1100 different components from Escherichia coli and should be capable of resolving a maximum of 5000 proteins. A protein containing as little as one disintegration per min of either 14C or 35S can be detected by autoradiography. A protein which constitutes 10 minus 4 to 10 minus 5% of the total protein can be detected and quantified by autoradiography. The reproducibility of the separation is sufficient to permit each spot on one separation to be matched with a spot on a different separation. This technique provides a method for estimation (at the described sensitivities) of the number of proteins made by any biological system. This system can resolve proteins differing in a single charge and consequently can be used in the analysis of in vivo modifications resulting in a change in charge. Proteins whose charge is changed by missense mutations can be identified. A detailed description of the methods as well as the characteristics of this system are presented.
TL;DR: This paper describes an alternate procedure for the first dimension which, unlike isoelectric focusing, resolves basic as well as acidic proteins, and involves a short time of electrophoresis toward the cathode and separates most proteins according to their isoelection points.
Abstract: A previously described two-dimensional electrophoresis procedure (O'Farrell, 1975) combined isoelectric focusing and sodium dodecylsulfate slab gel electrophoresis to give high resolution of proteins with isoelectric points in the range of pH 4–7. This paper describes an alternate procedure for the first dimension which, unlike isoelectric focusing, resolves basic as well as acidic proteins. This method, referred to as nonequilibrium pH gradient electrophoresis (NEPHGE), involves a short time of electrophoresis toward the cathode and separates most proteins according to their isoelectric points. Ampholines of different pH ranges are used to optimize separation of proteins with different isoelectric points. The method is applied to the resolution of basic proteins with pH 7–10 Ampholines, and to the resolution of total cellular proteins with pH 3.5–10 Ampholines. Histones and ribosomal proteins can be readily resolved even though most have isoelectric points beyond the maximum pH attained in these gels. The separation obtained by NEPHGE with pH 3.5–10 Ampholines was compared to that obtained when isoelectric focusing was used in the first dimension. The protein spot size and resolution are similar (each method resolving more than 1000 proteins), but there is less resolution of acidic proteins in this NEPHGE gel due to compression of the pattern. On the other hand, NEPHGE gels extend the range of analysis to include the 15–30% of the proteins which are excluded from isoelectric focusing gels. The distribution of cell proteins according to isoelectric point and molecular weight for a procaryote (E. coli) was compared to that of a eucaryote (African green monkey kidney); the eucaryotic cell proteins are, on the average, larger and more basic.
TL;DR: Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin, which results in a proportional gain in the inhibiting activity.
Abstract: A study has been made of the general properties of crystalline soybean trypsin inhibitor. The soy inhibitor is a stable protein of the globulin type of a molecular weight of about 24,000. Its isoelectric point is at pH 4.5. It inhibits the proteolytic action approximately of an equal weight of crystalline trypsin by combining with trypsin to form a stable compound. Chymotrypsin is only slightly inhibited by soy inhibitor. The reaction between chymotrypsin and the soy inhibitor consists in the formation of a reversibly dissociable compound. The inhibitor has no effect on pepsin. The inhibiting action of the soybean inhibitor is associated with the native state of the protein molecule. Denaturation of the soy protein by heat or acid or alkali brings about a proportional decrease in its inhibiting action on trypsin. Reversal of denaturation results in a proportional gain in the inhibiting activity. Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin. Methods are given for measuring trypsin and inhibitor activity and also protein concentration with the aid of spectrophotometric density measurements at 280 mmicro.
TL;DR: The current 2‐DE/MS workflow is described including the following topics: sample preparation, protein solubilization, and prefractionation; protein separation by 1‐DE with IPGs; protein detection and quantitation; computer assisted analysis of 2-DE patterns; protein identification and characterization by MS; two‐dimensional protein databases.
Abstract: Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. In spite of promising alternative or complementary technologies (e.g. multidimensional protein identification technology, stable isotope labelling, protein or antibody arrays) that have emerged recently, 2-DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. 2-DE enables the separation of complex mixtures of proteins according to isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or post-translational modifications. This is in contrast to liquid chromatography-tandem mass spectrometry based methods, which perform analysis on peptides, where Mr and pI information is lost, and where stable isotope labelling is required for quantitative analysis. Today's 2-DE technology with IPGs (Gorg et al., Electrophoresis 2000, 21, 1037-1053), has overcome the former limitations of carrier ampholyte based 2-DE (O'Farrell, J. Biol. Chem. 1975, 250, 4007-4021) with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5-12 has enabled the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow-overlapping IPGs provide increased resolution (delta pI = 0.001) and, in combination with prefractionation methods, the detection of low abundance proteins. Depending on the gel size and pH gradient used, 2-DE can resolve more than 5000 proteins simultaneously (approximately 2000 proteins routinely), and detect and quantify < 1 ng of protein per spot. In this article we describe the current 2-DE/MS workflow including the following topics: sample preparation, protein solubilization, and prefractionation; protein separation by 2-DE with IPGs; protein detection and quantitation; computer assisted analysis of 2-DE patterns; protein identification and characterization by MS; two-dimensional protein databases.
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