Sodium dodecylsulfate polyacrylamide electrophoresis

First Dimension Optimization After the second-dimension separation has been developed, the first-dimension flow rate is determined. This includes selecting a first-dimension column diameter to work at the flow rate selected. We illustrate the selection process with an application that addresses a column method for proteins that functions as a replacement for planar 2D gel electrophoresis (2DGE) within a narrow molecular weight and p/range. In the planar experiment, isoelectric focusing is performed in the first dimension and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS/PAGE) in the second dimension. 

Fig. 1.2 Protein blot analysis of human therapeutic protease inhibitor (HTPI) produced in alfalfa cell cultures using different promoters and subcellular targeting peptides as shown. Equal amounts of total soluble proteins from cell cultures were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinyldifluoride (PVDF) membrane. Monoclonal anti-HTPI IgGs were used for detection.

The basic material in seeds that is extractable with trichloroacetic acid solutions is ascribed to nonprotein nitrogen when the acid is in the 0.4-1.0 M concentration range. Gel electrophoresis on a sodium dodecylsulfate polyacrylamide medium pointed to the presence of 12 kDa polypeptides in soybean meal and 7, 10, 12 and 28 kDa in almond meal332. 

Parekh, B. S, Mehta, H. B., West, M. D., and Montelaro, R. C. (1985) Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis Anal Biochem 148, 87-92... 

Index Entries Recombinant green fluorescent protein GFPuv hydro-phobic interaction chromatography sodium dodecylsulfate polyacrylamide gel electrophoresis three-phase partitioning extraction. 

Jann, B., Reske, K., Jann, K. Heterogeneity of lipopolysaccharides. Analysis of polysaccharide chain lengths by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Eur J Biochem 60 (1975) 239-246. 

Capillary SDS-gel electrophoresis is a rapid automated separation and characterization technique for protein molecules and is contemplated as a modern instrumental approach to sodium dodecylsulfate-polyacrylamide slab-gel electrophoresis (SDS-PAGE). Size separation of SDS-protein complexes can be readily attained in coated capillaries filled with cross-linked gels or non-cross-linked polymer networks. Figure 9 depicts one of the early applications of the technique for the analysis of a standard protein test mixture ranging in size from 14.2 to 205 kDa. 

Apply the concentrated component II to a column of Sephacryl S-300 (2.5x95 cm, Pharmacia Biotech), which is equilibrated with 50 mM PB, pH 7.5, and elute with the buffer. Collect the fractions containing component II and concentrate by ultrafiltration. At this stage, check the purity of the component by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). If the concentrated fraction still contains any impurities, repeat the gel filtration under the same conditions. 

MTs = metallothioneins NMR = nuclear magnetic resonance pKa = negative logarithm of acid dissociation constant PMT = photomultiplier tube QITMS = quadrupole ion trap mass spectrometry SBSE = stir bar sorptive extraction SDS-PAGE = sodium dodecylsulfate polyacrylamide gel electrophoresis Se-Cis = selenocystine Se-Cys = selenocysteine Se-Et = selenoethionine Se-Hcy = seleno-homocysteine Se-Met = selenomethionine SEC = supercritical fluid chromatography TBT = tributyltin TETRA = tetramethylarsonium ion Tf = transferrin TMAsO = trimethylarsineoxide TMSe = trimethylselonium ion TOFMS = time of flight mass spectrometry USN = ultrasonic nebulizer. 

A number of separation modes are possible. In native gel electrophoresis, charged analytes are distinguished according to their apparent mobility, Papp, and size. In sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analytes are treated such that they all exhibit the same charge to size ratio. Hence, they are separated only by differences in their size. This method can be used for... 

El. Erickson, L. A., Lawrence, D. A., and Loskutoff, D. J., Reverse fibrin autography A method to detect and partially characterize protease inhibitors after sodium dodecylsulfate-polyacrylamide gel electrophoresis. Anal. Biochem. 137, 454 63 (1984). 

Schagger, H., and Jagow, G. (1987). Tricine-sodium-dodecylsulfate Polyacrylamide Gel Electrophoresis for the Separation of Proteins in the Range from 1-100 kDa, Anal. Biochem. 166 368-379. 

Prussak, C. E., et al. (1989). Peptide Production from Proteins Separated by Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis, Anal. Biochem. 178 233-238. 

Fig. 1. Ultrogel AcA 54 chromatography of sARF I effect of dimyristoyl phosphatidylcholine (DMPC) on ADP-ribosylation of Gsa and choleragen A protein. Purified sARF I was chromatographed on a column (1.2 x 104 cm) of Ultrogel AcA 54. Fractions (1 ml) were collected and samples were (A) analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assayed for ARF activity in a reaction mixture containing Gg (0.4 jig), choleragen (25 pg), and 100 pM GTP without (B) or with (C) 1 mM DMPC. (A) SDS-PAGE of 250 pi of fraction plus bovine serum albumin, 10 pg. Lanes 1-4, fractions 68, 72, 76 and 80 Lane 5, standard proteins, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, a-lactalbumin. (B) and (C) Autoradiograms of ADP-ribosylated proteins. Lanes 1-4, fractions 68,72,76 and 80 Lane 5, column buffer.

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