Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate.

Quantifying Absolute Protein Synthesis Rates Reveals Principles Underlying Allocation of Cellular Resources

Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea

Polyacrylamide gel electrophoresis (PAGE) provides a versatile, gentle, high resolution method for fractionation and physical-chemical characterization of molecules on the basis of size, conformation, and net charge. The polymerization reaction can be rigorously controlled to provide uniform gels of reproducible, measurable pore size over a wide range. This makes it possible to obtain reproducible relative mobility (Rf) values as physical-chemical constants. Application and extension of Ogston9s (random fiber) model for a gel allows for calculation of molecular volume, surface area, or radius, free mobility, and valence from RJ measurements at several gel concentrations, to calculate gel concentration for optimal resolution, and to predict behavior of macromolecules on gel gradients by computerized methods. Extension of classical moving boundary theory has been used to generate multiphasic buffer systems (providing selective stacking, unstacking, restacking, and preparative steady-state-stacking) with known operating characteristics for any pH at 0° and 25°C. A general strategy for isolation of macromolecules and for macromolecular mapping has been developed. Preparative scale PAGE is operational for milligram loads and feasible for gram quantities.

https://science.sciencemag.org/content/sci/172/3982/440.full.pdf

Polyacrylamide Gel Electrophoresis

Estimation of molecular radius, free mobility, and valence using polyacrylamide gel electrophoresis☆

Molecular weight estimation of polypeptides by SDS-polyacrylamide gel electrophoresis: further data concerning resolving power and general considerations.

Polyribosonmes have been shown to be the active uiiit of proteirn synlthesis in gamma globulin-producing cells from both rabbit lymph nodes1 andl mouse plasmna cell tumors.2 7S gamma globulii conisists of two heavy (H) anid two light (L) chains linked by disulfide bonds. The availability of techniques for separating and identifying the two chains affords a unique opportunity to study the synthesis and assembly of the polypeptide chains of a disulfide-linked protein. A/louse plasma cell tumors provide a cell population in which 20-30 per cent of the newly made protein is homogeneous gamma globulin. With these tunmor cells it has been possible to determine (1) whether the two chains are made on polyribosomes of different sizes, (2) whether the two chains are assembled on polyribosomes, (3) the relative rates of synthesis of the two chains, and (4) the approxinmate size of the messenger RNA's. AMaterials and Methods.-Two transplaintable inouise plasmna cell tumnors wer e used. Cells from the MPC-11 tumor (provided by Dr. John Fahey) produce approximately four tirnes as many L as H chains. The B-J tumor (provided by Dr. Elliot Osserman) synthesizes L chains, but few if any H chains. Immunologic an-d electrophoretic analysis indicated that these two polypeptides represented 20-30% of the total protein synthesized by both tumors. Cells were teased from the tumors, filtered through a stainless steel screen, washed four times, and resuspended in Eagle's medium4 containing I/loo the normal amounts of amino acids.3 After adjusting the cell concentration to 1-2 X 107 cells per ml and preincubating at 370 for 15 min, the cells were exposed to various mixtures of C14-amino acids at 10-20 mc/ml. Incorporation of the radioactive precursors was stopped by adding 10 vol of chilled Earle's saline, after which 107 cells were washed three times in cold Earle's saline and resuspended in 1 ml of a cytoplasmic extract freshly prepared from 108 HeLa cells.' The tumor cells were then disrupted by adding desoxycholate to a final conceltration of 0.5%, the cell lysates were immediately layered on 15-30% linear sucrose gradients,6 and centrifuged for 130-150 min at 24,000 rpm in the SW25.1 swingingbucket rotor (Spinco). Gradients were analyzed for UV absorbancy at 26() mn, anid for acid-precipitable radioactivity as previously described.3 "Pulse-chase" experiments were performed by iticubatiiig cells for 11/2 tllill withb a inxtUil't' of C14-labeled argininie, lysine, threonine, valinie, and leulcine (New Ei-iglaiid Niiclear Corp., 150)30()0 me/mM) at a final coticentratioln of 1l0-20 ,uc/ml an-id "chasing'' with a 200-fold excess of tnlabeled amin-o acids. Samples takeii at the time of additioni of the chase, an-d at 15-sec iiitervals thereafter, were analyzed for polyribosome-associated acid-precipitable radioactivity. The addition of the uinlabeled amino acids immediately stopped the incorporation of radioactive label into acid-precipitable material. The identification and quantification of H and L chains (a) in whole-cell lysates, (b) as newly completed and released polypeptide chains from the tops of sucrose gradients, and (c) as nascent polypeptides on polyribosomes, was carried out by a modification of polyacrylamide gel electrophoresis.7 Samples were prepared for electrophoresis by treatment with 10% acetic acid, 1% sodium dodecyl sulfate (SDS), 0.5 M urea, and 1% 2-mercaptoethanol (ME). Under these conditions, aggregated or insoluble proteins are solubilized so that they are neither lost nor trapped at the origin of the gel.7' 8 The sample was thern dialyzed at room temperature for 16 hr against, 0.1% SDS, 0.5 M urea, 0.1.1yo AME, an-d 0.01 M phosphate btiffer at pll 7.1. Five per cent polyacrylamlide gels were plepared inl the samne blLff er btit withl .1 phosphate coticeiitlat ioii of 0.1 AE,

https://www.jstor.org/stable/pdfplus/57393.pdf

Jacob V. Maizel

Papers

Molecular weight estimation of polypeptides by SDS-polyacrylamide gel electrophoresis: further data concerning resolving power and general considerations.

Polyribosomal synthesis and assembly of the H and L chains of gamma globulin.

Synthesis of excess light chains of gamma globulin by rabbit lymph node cells.