Sodium dodecyl sulfate polyacrylamide gel electrophoresis for direct quantitation of protein adsorption

A simple method, sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with direct protein adsorption analysis (SDS–PAGE/DPA), is presented here for the quantitation of adsorption-caused protein loss. No complicated steps and expensive equipment are involved, and this method is capable of measuring proteins adsorbed on sample vials at extremely low concentrations (in pg/μl). We used this method to characterize the effects of concentration, time, and volume on adsorption. We also applied this method to discover differential sample loss in protein mixtures and its utility in developing preventive strategies of adsorption.     

Sodium dodecyl sulfate-agarose gel electrophoresis for the detection and isolation of amyloid curli fibers

Curli are amyloid-like fibers on the surface of some strains of Escherichia coli and Salmonella enteritidis. We tested the use of horizontal sodium dodecyl sulfate (SDS)–agarose gel electrophoresis to detect, isolate, and quantitate curli. Cell extracts fractionated in SDS–agarose gels and stained with Coomassie blue exhibited a soluble fraction that entered the gel and an insoluble fraction that remained in the well. Much more insoluble material was observed with curli-proficient strains than with strains that do not make curli. Both highly purified curli and the insoluble material isolated from an SDS–agarose gel could be dissociated into monomers when treated with formic acid. For quantitation, we immobilized samples in SDS–agarose prior to electrophoresis. This avoids losses during the staining of the gel. Our methods provide a rapid and simple fractionation of curli using equipment that is readily available.     

The nature of the light-harvesting complex as defined by sodium dodecyl sulfate polyacrylamide gel electrophoresis

Reelectrophoresis of the oligomer form (CP II1) of the chlorophyll $\text{a}\text{b}$ light-harvesting complex (LHC) from the green alga Acetabularia yields two green bands which run at the position typical of the monomer (CP II). The upper green band (CP II₁) is enriched in the 27 kDa polypeptide of the LHC, while the lower is enriched in the 26 kDa polypeptide. The fact that both bands have both chlorophyll (Chl) a and b, and in the same ratio, implies that the LHC is made up of two Chl $\text{a}\text{b}$ proteins. Neither of these bands can be attributed to the Chl $\text{a}\text{b}$ complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432). Resolution of CP II₁ and CP II₂ of spinach can be obtained if sucrose gradient fractions of an octylglucoside extract are subjected to SDS-polyacrylamide gel electrophoresis. CP II₁ and CP II₂ are interpreted as being fundamental subunits of the light-harvesting complex as it is defined on SDS-polyacrylamide gels.     

A method for in-gel fluorescent visualization of proteins after native and sodium dodecyl sulfate polyacrylamide gel electrophoresis

We have developed a simple one-step 30-min method for fluorescent visualization of proteins in native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) gels. The method is based on formation of strong fluorophores via potassium ferricyanide-provoked oxidation of tryptophan (Trp). Following PAGE, gels are soaked in water solution of potassium ferricyanide (100 mM) and NaOH (1 M) and are kept in the dark for 30 min. Gels are then transferred to water and scanned. The sensitivity of the method was slightly lower compared with standard Coomassie Brilliant Blue (CBB) staining. The method can be useful when rapid acquisition of data is of the essence. After preview, gels can be post-stained using the CBB protocol for further analysis. The intensity of fluorescence is dependent on Trp number, so the protocol might find application in the quantification of Trp residues as illustrated here. Importantly, there is room for improvement of the method. Namely, according to excitation–emission matrix analysis of stained protein bands, maximal fluorescence intensity (at 345/460 nm) was 3.5-fold higher compared with the settings that were available on a commercial imager (395/525 nm). As a supplement, we present an upgrade of the previously described method for in-gel detection of non-heme iron-binding proteins that also employs potassium ferricyanide.     

Immunological identification of complex proteins resolved by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis.

Immunological identification of an antigen resolved from a protein complex by sodium dodecyl sulfate polyacrylamide gel electrophoresis has been attained. The identification is based on the formation of immunoprecipitin lines after the antigen diffuses laterally from acrylamide gel transverse slices into a surrounding agarose gel. This technique was designed for study of contractile and regulatory protein complexes of non-muscle cells where the scarcity of tissue precludes easy purification or high yield of muscle-like proteins. It complements double-gel immunodiffusion or immunoelectrophoresis and its use may be extended to other protein complexes.     

Immunochemical reactivity of simian sarcoma virus polypeptides isolated by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis

The polypeptides associated with a zonal centrifugation purified simian sarcoma virus propagated in lymphoblastoid NC-37 cells were isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) using a procedure designed to minimize the loss of immunochemical reactivity. The proteins p10, p15, p28, p36, p44, p75, and p86 were obtained in large yield and high degree of homogeneity. The electrophoretically purified p28 was analyzed by competition radioimmunoassay using antiserum to a pore exclusion and ion exchange purified simian sarcoma virus p28. Complete competition was observed with extracts of simian sarcoma virus infected cells. No competition was observed with uninfected or unrelated, infected cell extracts. The antigen-antibody affinity as measured by the slope of the competition curve using antiserum to p28 and 125I-labeled and electrophoretically purified p28 was the same as that for the p28 released from sonication-disrupted simian sarcoma virus. The data indicates that preparative purifications by polyacrylamide gel electrophoresis in the presence of SDS may be generally applicable for the isolation of proteins with essentially the same immunospecificities and affinity for a specific antiserum as proteins isolated by procedures that avoid the use of SDS and electrophoresis.     

Dispersal of proteolipid macroaggregates with trifluoroacetic acid and analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis

The propensity of highly purified proteolipids to form macroaggregates in aqueous solutions, especially when heated with sodium dodecyl sulfate (SDS), with or without thiol reagents, has made qualitative and quantitative analyses of individual species by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) difficult and unreliable. Comparisons of proteolipid profiles from liver, brain, and cultured human keratinocytes demonstrate that 40-72% of the total proteolipid in SDS-PAGE sample buffer is in the form of macroaggregates. Treatment of proteolipids with neat trifluoroacetic acid (TFA) followed by removal of the TFA and incubation in cold SDS-PAGE sample buffer causes complete dispersal of the macroaggregates and allows recovery of virtually all of the proteolipid applied to gels (increasing yields by as much as 3.6 times, depending on tissue type). Gels of TFA-treated samples display differences not only in the relative amounts of individual species but also in novel species not found in untreated samples. Eluted macroaggregates treated with TFA display the same SDS-PAGE banding profiles as TFA-treated whole proteolipids. Hence, routine TFA treatment of proteolipids prior to SDS-PAGE increases total proteolipid yields, allows reliable quantitation of individual apoprotein species, and reveals species previously obscured by the formation of macroaggregates.     

Prefractionation of protein samples for proteome analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

To isolate high molecular weight (HMW) or low-abundance proteins we exploited the high resolving power provided by the molecular sieves of polyacrylamide gel matrices. Rice-leaf protein extracts were applied to a single well of an SDS-polyacrylamide gel with prestained molecular size markers at both ends. After electrophoresis, the gel was cut into 4 segments according to size, and each segment was ground in extraction buffer. The eluted proteins were separated from the gel matrix by centrifugation followed by acetone precipitation, and the precipitated proteins were subjected to SDS-PAGE and 2-DE. The SDS-PAGE-based prefractionation method provided non-overlapping discrete sample pools. About 27% more protein spots were detected in the fractionated samples than in the unfractionated samples, and 17% were enhanced. The improvement was especially prominent in the case of HMW proteins. Well-separated HMW proteins were analyzed by MALDI-TOF mass spectrometry. The molecular masses of the identified proteins in the > 48 kDa gel segment were distributed between 50 and 112 kDa, thus validating this prefractionation method. Identified HMW proteins with similar mass but different pI were mostly isoforms. Thus SDS-PAGE-based size prefractionation provides improved separation and detection of HMW proteins.     

Detection of histones by DNA binding ability after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate

Histones can be detected on the basis of their binding to DNA after electrophoresis in sodium dodecyl sulfate-polyacrylamide gels containing DNA. The method depends on the ability of individual histone components to 1) renature, 2) bind to DNA, and 3) prevent ethidium bromide binding and fluorescence. This technique can provide qualitative and, possibly, quantitative information on histones in crude extracts.     

检测SDS-聚丙烯酰胺凝胶电泳中家蝇蛋白的新方法——海波银染法

介绍一种检测SDS聚丙烯酰胺凝胶电泳中家蝇幼虫蛋白的新方法-海波银染法。该方法对传统银染方法中的试剂与步骤加以改进,省略了乙醇固定与洗涤步骤,只需20 min即可完成全部染色过程,且仅在国产分析纯试剂及普通操作条件下,灵敏度可达毫微克级水平。     

Molecular weight determinations of proteins by polyacrylamide gel electrophoresis with sodium dodecyl sulfate in just the sample solution

This report describes a simple modification of the procedure for SDS polyacrylamide gel electrophoresis by using SDS in the sample solution and eliminating its use in the gel and the electrode compartments. The results obtained were comparable to those with SDS in the entire system. The log molecular weight-relative mobility plots for a variety of proteins, the reproducibility and the appearance of the bands such as their intentisy and sharpness were very similar. Therefore SDS in the entire system is not necessary to determine the molecular weight of a protein.     

Reliability of molecular weight determination of proteins by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate

Thirty-four proteins with molecular weights (MWs) between 1.3 × 10⁴ and 9.5 × 10⁵ and seven polypeptide chains of some of these proteins were incubated in sodium dodecyl sulfate and submitted to electrophoresis in a polyacrylamide gradient gel (3 to 30%; relative percentage of bisacrylamide: 8.4%). Analysis of the results confirmed the reliability of the relationship: log(MW) = alog(T) + b, where T is the polyacrylamide concentration reached by a protein, a is the slope, and b is the Y intercept of the regression line. In fact the mean deviation between the expected and the observed MW was ±5.5%. In addition we have shown that MWs of unreduced and reduced proteins can be estimated with the same accuracy and tha MWs of glycoproteins can also be determined with the same accuracy as other proteins.     

Use of capillary electrophoresis-sodium dodecyl sulfate to monitor disulfide scrambled forms of an Fc fusion protein during purification process

Overexpression of recombinant Fc fusion proteins in Escherichia coli frequently results in the production of inclusion bodies that are subsequently used to produce fully functional protein by an in vitro refolding process. During the refolding step, misfolded proteins such as disulfide scrambled forms can be formed, and purification steps are used to remove these product-related impurities to produce highly purified therapeutic proteins. A variety of analytical methods are commonly used to monitor protein variants throughout the purification process. Capillary electrophoresis (CE)-based techniques are gaining popularity for such applications. In this work, we used a nonreduced capillary electrophoresis–sodium dodecyl sulfate (nrCE–SDS) method for the analysis of disulfide scrambled forms in a fusion protein. Under denatured nonreduced conditions, an extra post-shoulder peak was observed at all purification steps. Detailed characterization revealed that the peak was related to the disulfide scrambled forms and was isobaric with the correctly folded product. In addition, when sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was used during the CE–SDS peak characterization, we observed that the migration order of scrambled forms is reversed on CE–SDS versus SDS–PAGE. This illustrates the importance of establishing proper correlation of these two techniques when they are used interchangeably to guide the purification process and to characterize proteins.     

The isolation of immunogenic molecular entities from immunogenic and nonimmunogenic tumor homogenates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Summary YAC, a Moloney-virus-induced tumor of A-strain mice, is a nonimmunogenic tumor. Mice injected with the inactivated neoplastic cells and challenged with viable tumor cells did not survive longer than mice that received the challenge dose alone. The homogenate of this nonimmunogenic tumor was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the gel slices containing isolated molecular entities were injected into various groups of mice. The mice were challenged with low doses of viable tumor cells (10–30 cells) and their survival time was recorded. Small but significant numbers of mice injected with apparent 80–90 K SDS-PAGE-isolated molecular entity rejected the tumor or survived longer than the control groups of mice. Spleen cells from mice injected with 80–90 K molecular entity inhibited the YAC tumor cotransferred with them to naive recipients (Winn assay). Spleen cells from mice injected with monoclonal antibody against nonspecific T-cell helper factor and immunized with 80–90 K SDS-PAGE-isolated molecular entity failed to inhibit the tumor growth in naive recipients, indicating that helper T cells are involved in induction of the antitumor resistance. Nylon-wool-passed splenocytes from mice injected with 80–90 K inhibited tumor growth in some of the recipient mice. Spleen cells from these mice treated with anti-Thy-1 and complement also inhibited the tumor growth in some of the recipients, suggesting that the effector cells were both T and non-T cells. C57BL/6 mice immunized with apparent 20 K SDS-PAGE-isolated molecular entity of RBL5 tumor also induced in vivo resistance to the syngeneic viable RBL5 cells, but not to the syngeneic B16 melanoma cells, indicating the specificity of the protective effect. The practical and theoretical implications of these findings are discussed.     

Use of capillary sodium dodecyl sulfate gel electrophoresis to detect the prion protein extracted from scrapie-infected sheep

Scrapie in sheep and in goats is the prototype of a group of transmissible spongiform encephalopathies (TSE). A feature of these diseases is the accumulation in the brain of rod shaped fibrils that form from an aggregated protein that is a protease-resistant form of a modified normal host cell protein. In this study, we compared SDS gel capillary electrophoresis to conventional SDS-PAGE and Western blot to detect the monomer of this aggregated protein. This prion protein was extracted from the sheep brain by homogenizing the brain stem (10%, w/v) in 0.32 M sucrose and by using a series of ultracentrifugation steps and treatment with sodium lauroyl sarcosine and proteinase K. After the final centrifucation step, the pellet was resuspended in 0.01 M Tris pH 7.4 in a volume equivalent to 0.1 ml/g of brain used. This resuspended pellet was treated with 1% SDS and 5% 2-mercaptoethanol and boiled for 10 min. The analysis was done in a Beckman P/ACE 5500 using a SDS gel capillary (eCap SDS14-200 Beckman capillary). In infected sheep brain samples, but not normal sheep, a major peak at a molecular mass of 16.1 kDa and a minor peak with a leading shoulder were observed. Since the molecular mass determined for this protein was lower than that estimated on Western blot (22.4 kDa), a Ferguson plot was made to determine if there were abberations in the molecular mass determination. After correction, the major peak was estimated to be 19.2 kDa. This has a better correlation with that determined by SDS-PAGE and Western blot. The equivalent amount of brain sample in the capillary was 50 μg. For Western blot, the amount of brain sample was 20 mg. For this assay, this is 100 times less than that needed for Western blot for sheep samples.     

A single-sample method for determination of carbohydrate and protein contents glycoprotein bands separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis.

A method is described for determination of carbohydrate and protein contents of glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Blots were stained, and appropriate pieces of PVDF membranes were excised, destained, and subjected to sequential hydrolysis with 0.2 M trifluoroacetic acid (TFA) for 1 h at 80 degrees C, then with 2 M TFA for 4 h at 100 degrees C, and finally with 6 M HCl at 100 degrees C for 24 h to release sialic acids, neutral sugars with hexosamines, and amino acids, respectively. In some instances preliminary methanolysis was used. Carbohydrates including sialic acids were quantitated by high pH anion exchange chromatography with pulsed amperometric detection. Protein content of the bands was determined as amino acids by the fluorescamine or ninhydrin method. In the calculation of results proper adjustments were made for small amounts of fucose released by hydrolysis with 0.2 M TFA at 80 degrees C, and for partial degradation of protein during hydrolysis with 2 M TFA at 100 degrees C. Recoveries of amino acids from hydrolysates of glycoproteins that had been electroblotted onto PVDF membranes equaled those of carbohydrates. This was possible because of preliminary hydrolysis of glycoproteins with TFA, as well as washing of wet, instead of dried, PVDF membranes after hydrolysis with 6 M HCl. The two modifications increased yields of amino acids by about 30%. The method was successfully applied to the determination of molar and weight percentage composition of human transferrin, band 3 protein, glycophorin A, and alpha(1)-acid glycoprotein. In each case the results obtained for directly hydrolyzed and electrophoresed/electroblotted glycoproteins were practically identical. We also determined the glucosamine content of band 4.1 protein of erythrocytes.     

Proteins and polypeptides of envelope membranes from spinach chloroplasts. I. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis separations

Polypeptides of spinach chloroplast envelopes were separated by electrophoresis in an SDS-polyacrylamide gradient gel. At least 37 polypeptides were resolved; nine were prominent. Two (M_r 54 000 and 16 000) were also found in the stroma fraction and identified by peptide mapping and isoelectric focusing in the second dimension as the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Proteins of the chloroplast envelope were also separated by isoelectric focusing. An adaptation of a previous method (Ames, G.F.L. and Nikaido, K. (1976) Biochemistry 15, 616ndash;623), using solubilization in SDS and isoelectric focusing in the presence of a high concentration of Nonidet P-40, gave the best separation and resolved the envelope membranes into at least 21 proteins. The major band (pI 6.85) contained both subunits of the carboxylase and at least two additional polypeptides which corresponded to the prominent bands found in SDS gel electrophoresis of chloroplast envelopes.