Isolation of protein components from rat lung lamellar bodies

Lamellar bodies isolated from 10% (w/v) rat lung homogenates by discontinuous sucrose gradient centrifugation were shown to contain variable amounts of adhering proteins. These contaminating proteins could be removed by either Sepharose 4B gel filtration or precipitation of the crude preparation at pH 11.5. Both purification methods yielded membrane preparations with a phospholipid-to-protein ratio of 10.0 μmol/mg. Nearly complete separation of lamellar body phospholipid and protein could be achieved upon application of the purified membranes to DEAE-cellulose in the presence of 0.2% (v/v) Triton X-100. Phospholipid analyses showed that 83% of total lipid phosphorus was recovered in phosphatidylcholine. In phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and phosphatidylinositol recoveries amounted to 4, 8, 2 and 2%, respectively. Molecular mass determinations of the isolated protein component of lamellar bodies by means of SDS polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue revealed the presence of three protein bands with molecular masses of 64, 33 and 31 kDa. Upon staining with silver a 16 kDa protein was also visible. Sephadex G-100 gel filtration showed only one protein peak corresponding to a molecular mass of 64 kDa when protein was assayed with Coomassie brilliant blue.     

A simple infrared spectroscopic method for the measurement of expired 13CO2.

A simple method for determination of proteins, initially solubilized in Tris buffer containing sodium dodecyl sulfate (SDS), mercaptoethanol, and sucrose, is described. This method is based on protein and Coomassie brilliant blue G-250 binding but it involves the removal of excess SDS by precipitation with 100 mm potassium phosphate buffer, pH 7.4 to 7.5, prior to protein determination. It has been established that the precipitation of excess SDS does not lead to the removal of the solubilized proteins. Therefore the method is applicable to both water-soluble and water-insoluble protein samples.     

Staining properties of bovine low molecular weight hydrophobic surfactant proteins after polyacrylamide gel electrophoresis.

Reports describing polyacrylamide gel electrophoresis patterns of bovine hydrophobic surfactant proteins are not consistent. In this study, we found unusual staining characteristics of these proteins that may explain some of these inconsistencies. Low molecular weight surfactant proteins extracted from bronchoalveolar lavage with organic solvent are partially delipidated with Sephadex LH-20 chromatography using chloroform and methanol. Fractions from the first protein peak are dried under nitrogen then subjected to SDS electrophoresis on 20% polyacrylamide gels. Under nonreducing conditions, silver staining identifies 5- and 26-kDa bands, and Coomassie blue identifies 6-, 12-, and 26-kDa bands. When gels are stained with Coomassie blue then silver, the 5- and 26-kDa bands stain with silver and 6- and 12-kDa bands remain stained with Coomassie blue. If gels are first stained with silver then Coomassie blue, similar results occur. We modified the silver staining protocol by treating gels with dithiothreitol or 2-mercaptoethanol after electrophoresis. With this modification, 5-, 6-, 12-, 26-, and also 17-kDa bands are identifiable. Using the modified protocol and restaining gels previously stained with silver, 6-, 12-, and 17-kDa bands that were not identified previously all became visible. In further experiments, protein bands of 6-, 12-, and 26-kDa that were identified by Coomassie blue were electroeluted under nonreducing conditions. After electrophoresis of the eluted 26-kDa protein, bands of 17-, and 26-kDa under nonreducing, and 8-kDa only under reducing conditions, were apparent by using the modified silver protocol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Imidazole-SDS-Zn reverse staining of proteins in gels containing or not SDS and microsequence of individual unmodified electroblotted proteins.

A reverse staining procedure is described for the detection of proteins in acrylamide and agarose gels with and without SDS. Protein detection occurs a few minutes after electrophoresis. The sensitivity on acrylamide gels is higher than that of Coomassie blue staining either on acrylamide gels or on electrotransferred membranes. Sequencing of protein bands only detected by reverse staining on the gel and not by Coomassie blue is demonstrated.     

Single-step electroelution of proteins from SDS-polyacrylamide gels and immobilization on diisothiocyanate-glass beads in prepacked capillary columns for solid-phase microsequencing

A new method for immobilization of proteins purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to sequencing is described. It utilizes a simple apparatus that permits the simultaneous electroelution of proteins from gel slices and attachment to diisothiocyanate-activated glass beads prepacked in capillary tubes [S-P. Liang and R. A. Laursen, Anal. Biochem. 188, 366-373 (1990)]. Transfer/attachment yields of greater than 80% within 90 min were observed for several 125I-labeled proteins with a range of molecular weights using 0.2 M sodium phosphate (pH 8.9) buffer containing 0.1% SDS. The method has the advantage of high capacity, relative simplicity, and insensitivity to the presence of SDS and Coomassie blue stain. The highest transfer yields were obtained when proteins were run on gels which had been aged for at least 12 h. For 100- to 1000-pmol samples, the sequenceable amount of protein, including transfer, was generally 30-60%, with an average repetitive yield of 95%. Factors which influence sample recovery and sequencing yield are discussed.     

Purification of protein from a crude mixture through SDS-PAGE transfer method

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) transfer method was used for purification and enrichment of the protein from crude sample. Coomassie bluc/ZnSO4 stained protein band(s) containing intact polyacrylamide gel were loaded on to another polyacrylamide gel either alone or as pooled gel bands. Two/three bands were combined together and arranged tightly over one another, sealed with stacking gel and ran in another gel, which was quite useful for enrichment and purification of a particular protein from a complex mixture. Recovery of protein by gel transfer method was found to be 70% in case of ZnSO4 staining, whereas around 30% recovery was possible, following Coomassie blue staining. The method described here for purification of protein(s) from a complex mixture, following gel transfer procedure could be useful for further characterization of the desired protein.     

Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein

We have developed a novel method for constructing synthetic genes that encode a series of peptides comprising perfect repeat motifs based on a high molecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed. A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Escherichia coli. Activity of the synthetic genes in E. coli was confirmed by Northern blot analysis but SDS-PAGE of crude protein extracts failed to show any expressed peptides when stained using Coomassie brilliant blue R250. However, Western blots probed with a HMW glutenin subunit-specific polyclonal antibody showed the presence of the R6 peptide (M(r) 22005) in the crude cell extracts and both this and the R3 peptide (M(r) 12005) were subsequently purified by extraction with hot aqueous ethanol followed by precipitation with acetone and separated by RP-HPLC. The R4 and R5 peptides were not purified. The purified R3 and R6 peptides absorbed Coomassie brilliant blue R250 or other protein stains only weakly and this was considered to account for their failure to be revealed by staining of separations of the crude protein extracts. Circular dichroism spectroscopy showed that both peptides had similar beta-turn rich structures similar to the repetitive sequences present in the whole HMW glutenin subunits. We conclude that expression of perfect repeat peptides in E. coli is a suitable system for the study of structure-function relationships in wheat gluten proteins and other highly repetitive proteins.     

One-step purification of proteins from chicken egg white using counter-current chromatography

Proteins present in chicken egg white are separated by counter-current chromatography (CCC) in one step using a cross-axis coil planet centrifuge (X-axis CPC). The separation was performed with an aqueous polymer two-phase system composed of 16% (w/w) poly(ethylene glycol) 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 1.0 ml/min. From about 20 g of the crude egg white solution, lysozyme, ovalbumin, and ovotransferrin were resolved within 5.5 h. Each component was identified by 12% SDS gel electrophoresis with Coomassie brilliant blue staining.     

A micromethod for the quantitation of cellular proteins in Percoll with the Coomassie brilliant blue dye-binding assay

A simple procedure for the determination of cellular proteins in Percoll-containing samples is described. Percoll precipitated when particulate proteins were solubilized by dilution of the samples in a NaOH-Triton X-100 mixture. After centrifugation at high speed (12,000g), the supernatant was assayed for proteins with the Coomassie brilliant blue dye-binding assay. With an automatic spectrophotometer, 50-microliter aliquots gave a linear response between 0 and 3 micrograms of bovine serum albumin. After a fivefold dilution in the alkali-detergent mixture, proteins in samples containing up to at least 60% Percoll can be accurately quantitated on a standard curve prepared in the absence of Percoll. Because the sensitivity of the assay was better than 100 ng, the procedure outlined in this paper can also be used as a general protein micromethod.     

Protein-blotting on Polybrene-coated glass-fiber sheets. A basis for acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on sodium dodecyl sulfate/polyacrylamide gel

A procedure has been developed which allows the immobilization on glass-fiber sheets coated with the polyquaternary amine, Polybrene, of proteins and protein fragments previously separated on sodium-dodecylsulfate-containing polyacrylamide gels. The transfer is carried out essentially as has been used for protein blotting on nitrocellulose membranes [Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl Acad. Sci. USA 76, 4350-4354], but is now used to determine the amino acid composition and partial sequence of the immobilized proteins. Protein transfer could be carried out after staining the proteins in the gels with Coomassie blue, by which immobilized proteins are visible as blue spots, or without previous staining, after which transferred proteins are detected as fluorescent spots following reaction with fluorescamine. The latter procedure was found to be more efficient and yielded binding capacities of +/- 20 micrograms/cm2. Fluorescamine detection was of equal or higher sensitivity than the classical Coomassie staining of proteins in the gel. Immobilized proteins could be hydrolyzed when still present on the glass fiber and reliable amino acid compositions were obtained for various reference proteins immobilized in less than 100 pmol quantities. In addition, and more importantly, glass-fiber-bound proteins could be subjected to the Edman degradation procedure by simply cutting out the area of the sheet carrying the immobilized protein and mounting the disc in the reaction chamber of the gas-phase sequenator. Results of this immobilization-sequencing technique are shown for immobilized myoglobin (1 nmol) and two proteolytic fragments of actin (+/- 80 pmol each) previously separated on a sodium-dodecylsulfate-containing gel.     

Characterization of a protein from Trypanosoma cruzi trypomastigotes that cleaves non-immune IgG bound through its Fab fragment

A protein from Trypanosoma cruzi bloodstream trypomastigotes that binds IgG from man and several other animal species was isolated and characterized. The 52-kDa protein obtained from different T. cruzi cell extracts showed saturable binding with a K of 3.72 nM. Immunoblot analysis revealed that Fab, but not Fc, fragments of the Ig were recognized. When the protein was added to an unrelated C-fixation reaction, lysis was abolished in a dose-dependent fashion. When freshly prepared T. cruzi extracts were run in a 10% acrylamide SDS gel into which normal rabbit IgG was incorporated before polymerization, proteolytic activity, as seen by a transparent band after Coomassie blue staining, migrated in the same m.w. range of the 52-kDa protein. These data provide further clues to the mechanisms through which this pathogen escapes the host's immune response, thus maintaining a long-standing infection.     

A simple,rapid, and sensitive method for measuring protein concentration in subcellular membrane fractions prepared by sucrose density ultracentrifugation

The Multiskan spectrophotometric system and Coomassie brilliant blue G-250 protein-dye binding method have been used together to measure NaOH-solubilized protein in subcellular membrane fractions prepared from isolated rat adipose cells. Forty-eight samples can be read in duplicate within 1 min. Sucrose in concentrations up to 0.7 m interfere only moderately with the assay. A rapid and convenient method is, therefore, now available for multiple protein determinations following sucellular fractionation on sucrose gradients.     

Further evidence for the heterogeneity of serum albumin

Albumin samples from three species (avian, bovine and human) were electrophoresed on gradient polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS-PAGE). The resulting electrophoregram from each sample of serum albumin investigated showed multiple protein bands of a wide range of molecular weights. All seven samples of human serum albumin were found, using gel immunodiffusion, to be contaminated with other proteins. All but one sample was contaminated with proteins such as haptoglobin, alpha 1-glycoprotein, alpha 1-trypsin inhibitor, and prealbumin. This contamination accounts for part of the heterogeneity of these samples. Immunoblots, where the proteins were transferred to nitrocellulose and incubated with antisera, gave a better demonstration of the heterogeneity than Coomassie Blue staining and the immunoblotting procedure appeared to be more sensitive than the gel immunodiffusion technique. The heterogeneity of serum albumin demonstrated by the former technique included that of the monomer which was shown to be contaminated with antithrombin III. The commercial samples of human serum albumin, claimed as pure, were found to vary greatly in their tryptophan content, which also indicated heterogeneity. Heat treatment of human serum albumin with 1% SDS, followed by chromatography on agarose, removed the protein contaminants and with it the tryptophan. The presence of tryptophan in human serum albumin, therefore, indicated the presence of impurities.     

考马斯亮蓝染色双向电泳凝胶胶内酶切方法的改进

介绍一种简便高效的从考马斯亮蓝染色的双向电泳凝胶切取蛋白质点,通过胶内酶切制备基质辅助激光解吸电离飞行时间质谱(MALDI—TOF-MS)样品的方法。该方法操作简便,样品的质谱鉴定结果经网上数据库检索检出率可高达80％以上,适于我国目前小规模蛋白质组学研究的国情,便于推广应用。     

A simple, rapid, and sensitive procedure for the assay of endoproteases using Coomassie brilliant blue G-250

A rapid colorimetric method for the assay of proteolytic enzymes based on the binding of Coomassie brilliant blue G-250 to unhydrolyzed protein substrate is described. Considerable assay time is saved since the method does not require the separation of the hydrolyzed products from the undergraded protein substrate. The procedure is applicable to crude as well as purified preparations of various proteolytic enzymes and compares well with the procedure of M. L. Anson.     

Immunochemical Analysis of Triton X-100-Insoluble Residues from Micrococcus lysodeikticus Membranes

Triton X-100-insoluble residues from Micrococcus lysodeikticus membranes were analyzed by crossed immunoelectrophoresis after dispersal of the residues in sodium dodecyl sulfate (SDS). Conditions which produce no obvious distortion of the immunoprecipitate profile and which allow qualitative and quantitative analyses of the antigens present in the extracts are described. Two main antigens were detected; these were identified as succinate dehydrogenase (EC 1.3.99.1) and adenosine triphosphatase (EC 3.6.1.3). As determined by peak area estimations, the maximal release of succinate dehydrogenase and of adenosine triphosphatase from Triton X-100-insoluble membrane residues occurred at protein/SDS ratios of about 4.3:1 (0.2% SDS) and 6.8:1 (0.13% SDS), respectively. A comparison of enzyme activities of SDS extracts with those of untreated, control Triton X-100-insoluble membrane residues indicated that both the succinate dehydrogenase and the adenosine triphosphatase antigens were released with a full (or enhanced) catalytic potential at or below concentrations of SDS required to effect maximal solubilization of the enzyme in question. Evidence is also presented to suggest that the more acidic of the two components detected by crossed immunoelectrophoresis for the heterogeneous adenosine triphosphatase antigen is more sensitive to SDS than is the other. Both succinate dehydrogenase and adenosine triphosphatase lost catalytic activity and were denatured at protein/SDS ratios lower than 3.4:1.     

A method for quantitating nanogram amounts of soluble protein using the principle of silver binding

A highly sensitive and quantitative assay for measuring protein in solution based on the capacity of protein to bind silver is described. In this procedure, protein samples are first treated with glutaraldehyde and then exposed to ammoniacal silver. After 10 min, the reaction is terminated by the addition of sodium thiosulfate and the optical density measured at 420 nm. The useful range of the assay for the majority of standard proteins tested lies between 15 and 2000 ng. This represents a 100-fold increase in sensitivity over the Coomassie brilliant blue dye-binding procedure. There is little or no interference from carbohydrates, nonionic detergents, or ethanol, and pretreatment of protein samples with Bio-Gel P-2 to remove salts, thiol agents, EDTA, and sodium dodecyl sulfate makes this procedure compatible with most commonly used buffers. The cost in terms of silver utilization is nominal with a typical assay involving 10 samples tested in triplicate amounting to less than $0.02 U. S.     

Sericin from Bombyx mori cocoons. Part I: Extraction and physicochemical-biological characterization for biopharmaceutical applications

In the present research effort, production of crude sericin extracts from Bombyx mori silk cocoons was attempted using two different approaches. Sericin was extracted from cocoons by high-temperature autoclaving followed either by lyophilization or freezing-thawing precipitation, to obtain a crude sericin powder. The physico-chemical and biological characteristics of the crude sericin extracts were evaluated in detail, via FTIR, XRD, XRF, XRT, UV–vis scanning, TGA and DSC, protein quantification, antimicrobial activity, free radical scavenging activity, cytotoxic activity, potential for inducing chromosomal aberrations via Allium cepa assays, and genotoxicity via Comet™ analyses. The molecular weight distribution of the crude sericin extracts was also investigated, via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and the results duly compared to standard sericin. The results gathered clearly suggest that the crude sericin extracts had both obvious radical scavenging effects with the 2.2-diphenyl-1-picryl-hydrazil (DPPH) assay, and antibacterial activity, further suggesting that this protein might be a valuable addition for either food and biopharmaceutical applications.     

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.     

Immunoaffinity purification of Schistosoma mansoni soluble egg antigens

Schistosoma mansoni egg antigens were purified from a heterogeneous mixture of soluble egg antigens (crude SEA) with an immunoaffinity column that consisted of the specific anti-SEA antibodies contained in 16-week S. mansoni-infected mouse serum bound to Sepharose 4B. On sodium dodecyl sulfate (SDS) gel electrophoresis, the purified antigen fraction yielded at least eight bands staining with Coomassie blue and at least five bands staining with Coomaisse blue and at least five bands reacting with periodic acid-Schiff (PAS). All of the proteins in the antigenic fraction appear to contain carbohydrate residues. Upon immunoelectrophoresis the antigen yielded four precipitin arcs. The antigenic fraction isolated by means of the immunoaffinity column was then compared to various fractions obtained from concanavalin A (Con A) chromatography of SEA. The results of Ouchterlony immunodiffusion and immunoelectrophoresis indicate that the antigenic fraction isolated by immunoaffinity purification of SEA contains the major antigens found in the fractions obtained from Con A chromatography of SEA. The results of SDS gel electrophoresis indicate that the major PAS-reacting bands of the antigenic fraction isolated by immunoaffinity purification are found in the 3rd peak (bound fraction) resulting from Con A chromatography of SEA, whereas the major Coomaisse blue-staining band in the isolated antigenic fraction is found in the 2nd peak (unbound fraction) from Con A chromatography of SEA.