Urea SDS-PAGE Research Articles

An Intracellular Laccase Is Responsible for Epicatechin-Mediated Anthocyanin Degradation in Litchi Fruit Pericarp.

In contrast to the detailed molecular knowledge available on anthocyanin synthesis, little is known about its catabolism in plants. Litchi (Litchi chinensis) fruit lose their attractive red color soon after harvest. The mechanism leading to quick degradation of anthocyanins in the pericarp is not well understood. An anthocyanin degradation enzyme (ADE) was purified to homogeneity by sequential column chromatography, using partially purified anthocyanins from litchi pericarp as a substrate. The purified ADE, of 116 kD by urea SDS-PAGE, was identified as a laccase (ADE/LAC). The full-length complementary DNA encoding ADE/LAC was obtained, and a polyclonal antibody raised against a deduced peptide of the gene recognized the ADE protein. The anthocyanin degradation function of the gene was confirmed by its transient expression in tobacco (Nicotiana benthamiana) leaves. The highest ADE/LAC transcript abundance was in the pericarp in comparison with other tissues, and was about 1,000-fold higher than the polyphenol oxidase gene in the pericarp. Epicatechin was found to be the favorable substrate for the ADE/LAC. The dependence of anthocyanin degradation by the enzyme on the presence of epicatechin suggests an ADE/LAC epicatechin-coupled oxidation model. This model was supported by a dramatic decrease in epicatechin content in the pericarp parallel to anthocyanin degradation. Immunogold labeling transmission electron microscopy suggested that ADE/LAC is located mainly in the vacuole, with essential phenolic substances. ADE/LAC vacuolar localization, high expression levels in the pericarp, and high epicatechin-dependent anthocyanin degradation support its central role in pigment breakdown during pericarp browning.

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Troubleshooting forum. SDS-page. What is the best way to separate proteins of similar molecular weight? (Thread 33111).

BioTechniquesVol. 56, No. 3 Troubleshooting ForumOpen AccessSDS-PAGEKristie NyboKristie NyboSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114144AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail This month's question from the Molecular Biolog y Forums (online at molecularbiology.forums.biotechniques.com) comes from the “Protein Methods” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but do not represent endorsements by, or the opinions of, BioTechniques.Molecular Biology Techniques Q&AWhat is the best way to separate proteins of similar molecular weight? (Thread 33111)Q I need to separate 2 proteins with molecular weights of 86 kDa and 80 kDa; however, I haven't obtained a good separation yet, even on a 6% polyacrylamide gel. To make matters worse, the two proteins are isoforms, and there are no commercially available antibodies against either one.I'm considering using a 4% gel or maybe a 2-D gel, although both isoforms seem to have the same isoelectric point (pI). Can anyone recommend a better technique for separating these proteins?A You're going the wrong way with the gel concentration; you want to be more restrictive. Try a 10% polyacrylamide gel with a narrow range gradient, such as 10-12%. You might also add 4 M urea to the gel.Q I did try 10% polyacrylamide on several occasions but still didn't get good separation. I will try again, this time adding urea to the sample as you suggest.Will the urea compress the protein bands so that they are distinguishable from bands with similar molecular weights?A Remember, your samples are already denatured (SDS and reducing agent). I don't think the urea will further denature the sample, but urea does enhance charge separations and sharpens banding.You should add urea to the gel, not just to the the sample. If you want to add urea to the sample, add it after the heating and cooling steps.Q I tried a 4 M urea SDS-PAGE but saw fuzzy bands after probing. Could that be because I did not add urea to my samples? What amount of urea should I add?A It is best if the urea concentration in the sample and gel is the same, but you can use a higher concentration in the sample if necessary.Were your bands separated enough to distinguish between the isoforms?Fuzzy bands can be the result of glycosylation, diffusion (from standing around too long before fixing or transfer, or soaking too long in the transfer buffer), or insufficient stacking, among other reasons.Q I ran another 6 M urea, 10% polyacrylamide gel with 6 M urea-containing samples and saw rather compact bands; however, I think the fuzzy bands could be due to differences in protein expression levels. I'm using cells from lymphoid organs and cell lines with varying expression levels of my protein isoforms of interest. It looks like I will need to titrate the amount of protein loaded since the separation is still not clear.Is it a good idea to let the dye front run off and only retain the protein of interest?A That depends on what you want to show. If you are only interested in those two bands, then run the dye out of your gel. If you want reproducible results, run the gels for a specific number of volt-hours.Q Since the separation is still not clear, does it make sense to use a higher percentage of urea?A 8 M urea may improve the separation and make the bands sharper, but you may want to try a 12% gel, or even better, a 10-12% gradient gel.Q Is a 30 min incubation of the samples in 8 M urea sufficient?A Incubation with urea is not necessary. Also, be sure never to heat the urea; it will decompose and carbamylate your protein.Q Even with these modifications, I see only minimal separation. One problem might be that I am loading too much protein on the gel. I will try reducing the input to reduce the thickness of the band.A We saw improved resolution of our proteins using two electrode buffers instead of Laemmli buffer, along with running and stacking gel buffers.A A longer gel with proper stacking or a gradient should work without too much band broadening, especially if you add some urea to the gel.FiguresReferencesRelatedDetails Vol. 56, No. 3 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print March 2014 Information© 2014 Author(s)PDF download

Evaluation of the rheological, textural, microstructural and sensory properties of soy cheese spreads

In this work, different processes including lactic acid bacteria fermentation, glucono-δ-lactone (GDL) coagulation and enzymatic hydrolysis were applied to produce soy cheese spread without any milk solid and/or alkaline metal caseinates added. All the soy cheese spread samples were studied with respect to the differences in the textural properties – utilizing TPA and rheological behavior, besides their microstructural features and sensory assessment. In addition, urea–SDS-PAGE assay was used to investigate the proteolysis in enzymatic hydrolysis process. As a result, the soy cheese spread sample produced by the combined use of GDL and lactic acid bacteria fermentation together with enzymatic hydrolysis process, was observed with better spread-ability, more acceptable sensory features, and stable homogeneous structure than other samples.

Analysis of Tropomyosin (Tm) Isoforms in Heart Muscle by LC/MS and Western Blotting Demonstrates Previously Uncharacterised High-Level Expression of γ Tm

Heart muscle has been considered to contain predominantly α-Tm (TPM1 gene). However this has not been investigated with tools that can clearly distinguish all tropomyosin isoforms. We initially used LC/MS to characterise the isoforms present in Tm purified from human heart muscle. Mass spectroscopy of Tm purified from human donor heart showed two dominant species, a 32754 Da peak, corresponding to acetylated α-Tm and a 32867 Da peak. The second peak was putatively identified by its mass as γ-Tm. These identities were confirmed by comparison with pure recombinant α and γ-skeletal tropomyosin expressed in the baculovirus/sf9 system (supplied by Dr Kristen Nowak, University Western Australia). The novel Tm is slow-skeletal Tm from the TPM3 gene, also known as γ-skeletal tropomyosin, (theoretical mass 32859.0 Da). Only very low levels (<5%) of κ Tm or of phosphorylated Tm were detected. Analysis of samples from failing heart muscle and in myectomy samples showed significantly reduced γ-Tm levels.The identity of the isoforms characterised in human heart was confirmed by urea SDS-PAGE, which can separate the two species, and the use of isoform-specific antibodies. Using the pure recombinant Tms, we demonstrated that monoclonal antibody CG3 was specific for the γ isoform, CH291 was specific for the α isoform and CH1 did not distinguish between the two isoforms. Western blotting with these specific antibodies confirmed significant quantities of γ-Tm and α-Tm in both human and mouse hearts. In mice, the level of γ-Tm depends on the strain, being around 50% in many strains, but low in FVB/N mice.This discovery is functionally significant as in vitro motility assays using pure γ-Tm and α-Tm shows significant differences between the isoforms.

Generation of Aβ38 and Aβ42 Is Independently and Differentially Affected by Familial Alzheimer Disease-associated Presenilin Mutations and γ-Secretase Modulation

Alzheimer disease amyloid beta-peptide (Abeta) is generated via proteolytic processing of the beta-amyloid precursor protein by beta- and gamma-secretase. Gamma-secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal anti-inflammatory drugs, including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42-amino acid Abeta(42) and concomitantly increase the levels of the rather benign Abeta(38). Here we show that Abeta(42) and Abeta(38) generation occur independently from each other. The amount of Abeta(42) produced by cells expressing 10 different familial Alzheimer disease (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of gamma-secretase, appeared to correlate with the respective age of onset in patients. However, Abeta(38) levels did not show a negative correlation with the age of onset. Modulation of gamma-secretase activity by sulindac sulfide reduced Abeta(42) in the case of wild type PS1 and two FAD-associated PS1 mutations (M146L and A285V). The remaining eight PS1 FAD mutants showed either no reduction of Abeta(42) or only rather subtle effects. Strikingly, even the mutations that showed no effect on Abeta(42) levels allowed a robust increase of Abeta(38) upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase Abeta(42) and to decrease Abeta(38). For mutants that predominantly produce Abeta(42), the ability of fenofibrate to further increase Abeta(42) levels became diminished, whereas Abeta(38) levels were altered to varying extents for all mutants analyzed. Thus, we conclude that Abeta(38) and Abeta(42) production do not depend on each other. Using an independent non-steroidal anti-inflammatory drug derivative, we obtained similar results for PS1 as well as for PS2. These in vitro results were confirmed by in vivo experiments in transgenic mice expressing the PS2 N141I FAD mutant. Our findings therefore have strong implications on the selection of transgenic mouse models used for screening of the Abeta(42)-lowering capacity of gamma-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to gamma-secretase modulators.

Unique constitution of photosystem I with a novel subunit in the cyanobacterium Gloeobacter violaceus PCC 7421

Constitution of the photosystem I complex isolated from the cyanobacterium Gloeobacter violaceus PCC 7421 was investigated by tricine–urea–SDS–PAGE, followed by peptide mass fingerprinting or N-terminal sequencing. Eight subunits (PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaL and PsaM) were identified as predicted from the genome sequence. A novel subunit (PsaZ) was discovered, but PsaI, PsaJ, PsaK and PsaX were absent. PsaB has a C-terminal extension with 155 amino acids in addition to the conserved region and this domain is similar to the peptidoglycan-binding domain. These results suggest that PS I complexes of G. violaceus have unique structural properties.

Expression of the murine CB 2 cannabinoid receptor using a recombinant Semliki Forest virus

A recombinant Semliki Forest virus (SFV) RNA construct, SFV1-mCB 2 RNA, was employed for the high-level expression of the murine CB 2 (mCB 2) cannabinoid receptor in baby hamster kidney cells. Biosynthetic radiolabel incorporation studies in concert with urea-sodium dodecylsulfate–polyacrylamide gel electrophoresis (urea-SDS–PAGE) and western immunoblotting revealed that two major proteins of approximately 26 and 40 kDa were produced by the construct. The 40 kDa product, but not the 26 kDa product, was glycosylated as determined by 2-deoxy- d-glucose incorporation and peptide- N-glycosidase F digestion analysis. Assessment of [ 3 H ]CP55940 ([ 3 H ]-(−)- cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]- trans-4-(3-hydroxypropyl)cyclohexanol) binding data for membranes of cells transfected with SFV1-mCB 2 RNA indicated a K d of 0.35±0.04 nM and a B max of 24.4±2.7 pmol/mg. A rank order of binding affinities for cannabinoids, which paralleled that reported for native mCB 2 receptors, was observed. The CB 2 receptor-specific antagonist SR144528 ( N-[(1 S)-endo-1,3,3-trimethyl bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) blocked binding of [ 3 H ]CP55940, while the CB 1 receptor-specific antagonist SR141716A [ N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide hydrochloride] had a minimal effect. These results indicate that the recombinant receptor expressed from SFV1-mCB 2 RNA exhibits properties, including ligand binding features, that are consistent with those for the native mCB 2 receptor. However, the presence of both 26 and 40 kDa receptor species is consistent with alternative translation from two AUG start sites using the SFV1-mCB 2 RNA expression system.

Purification of Integral Outer-Membrane Protein OmpC, a Surface Antigen from Salmonella typhi for Structure–Function Studies: A Method Applicable to Enterobacterial Major Outer-Membrane Protein

Extraction of the outer-membrane porin, OmpC, from Salmonella typhi Ty21a was done by using a modified salt-extraction procedure. It was possible to extract only the major outer-membrane protein (OMP) from the crude membrane using this method. Aberrant lipopolysaccharide (LPS) production in the galE mutant Ty21a has resulted in more isoforms of OmpC and subsequently led to anomalous mobility in SDS-PAGE. The purity of the preparation was confirmed by denaturing urea SDS-PAGE and N-terminal sequencing. The major OMP extracts had LPS of both bound and free forms. The free form of LPS could be removed by gel filtration and the bound form, largely, was removed using ion-exchange chromatography and by passing through ultrafiltration devices. This method has been used to extract the native trimer of OmpC, the major OMP, in a large scale, for structure–function studies. S. typhi Ty21a OmpC preparation yielded reproducible diffraction-quality crystals. Extracts of porin from wild-type Escherichia coli HB101, grown under high osmolarity conditions, showed a single species of OMP on SDS-PAGE. This suggests the possible application of the method to other gram-negative bacterial porins.

The decrease in the short variant of gsalpha protein is associated with an increase in [3H]CGP12177 binding, [3H]ouabain binding and Na, K-ATPase activity in brown adipose tissue plasma membranes of cold-acclimated hamsters.

Sucrose density gradient purified plasma membranes isolated from brown adipose tissue of cold-acclimated hamsters (4-10 weeks at 0-4 degreesC) were analysed for the content of the short (GsalphaS) and long (GsalphaL) variants of Gsalpha protein (the alpha subunit of the stimulatory G protein) and compared with the membranes isolated from control animals. The relative ratio between the two variants (GsalphaS/GsalphaL) decreased from 0.48 to 0.24 (P<0.01). This result, obtained by electrophoretic resolution of membrane proteins by standard SDS-PAGE and an immunoblot analysis with an antiserum oriented against an internal sequence of Gsalpha, was verified by resolution on urea-containing gels and an antiserum oriented against the C-terminus decapeptide of Gsalpha. Under these conditions, the GsalphaS/GsalphaL ratio was decreased from 0.41 to 0.31 (P<0.05). The total amount of both isoforms (GsalphaS plus GsalphaL) decreased to 83% (P<0.05) or 68% (P<0.01) by standard or urea SDS-PAGE respectively. These data demonstrate that cold-acclimation of hamster brown adipose tissue is associated with preferential decrease in the plasma membrane density of the short variant of the Gsalpha protein.%This decrease was paralleled by an increase in the other plasma membrane constituents, [3H]CGP12177 binding sites, [3H]ouabain binding sites and Na,K-ATPase activity to 147%, 212% and 191% respectively.

Molecular and Biochemical Characterisation of HMW Glutenin Subunits fromT. tauschiiand the D Genome of Hexaploid Wheat

Abstract Electrophoretic (urea SDS–PAGE) and chromatographic (RP–HPLC) analysis was performed on 8 allelic variants of HMW glutenin subunits derived from Triticum tauschii and from the D genome of a hexaploid wheat species ( Triticum macha ) and hexaploid landraces. These subunits had been previously identified using SDS–PAGE. The characterisation revealed that subunits Dy10 t and Dy12 t from T. tauschii could be differentiated from their bread wheat counterparts using both urea SDS–PAGE and RP–HPLC. In the latter case, the T. tauschii y-type subunits were clearly more hydrophobic than the Dy type subunits of bread wheat. The characterisation also suggested that subunit Dx5 t , derived from two separate T. tauschii accessions, did not contain the extra cysteine residue characteristic of Dx5 from bread wheat. RFLP analysis of the genes encoding the HMW glutenin subunits of interest suggested that the absence of Dx-type HMW glutenins in two hexaploid landraces was due to lack of expression of their encoding genes. The relationship between Hin dIII DNA fragment size and protein subunit size, as measured by electrophoretic mobility, is examined and discussed. Finally, the solubility properties of a HMW protein designated T1 (derived from T. tauschii accession AUS 18913) suggested that it was not a HMW glutenin subunit as was previously thought. Further studies are needed to clarify the identity of this subunit.

Purification of the 11- and 5-kDa Antibacterial Polypeptides from Guinea Pig Neutrophils

It has been known that neutrophils contain various antimicrobial components in the granules, which contribute to the oxygen-independent host defense mechanism. In this study, we have isolated the two antimicrobial polypeptides from guinea pig neutrophil granules. Urea–SDS–PAGE analysis revealed that the molecular masses of the polypeptides were 11 and 5 kDa under nonreducing conditions. Under reducing conditions, the molecular mass of the 5-kDa polypeptide did not change, whereas the molecular mass of the 11-kDa polypeptide changed to about 5 kDa, suggesting that the 11-kDa polypeptide is a dimer composed of 5-kDa subunits joined with a disulfide bond. The amino acid composition and sequence data indicated that the 5-kDa subunit of the 11-kDa polypeptide contained 9 lysine, 8 arginine, and 1 cysteine residues and that the 11-kDa polypeptide was a homodimer of G1LRKKFRKTRKRIQKLGRKIGKTGRKVWK AWREYGQIPYPCRI43(4599 Da) joined with one disulfide bond. Amino acid sequence of the 11-kDa polypeptide showed partial homology (19–30%) to the active peptides of rabbit and human cationic antimicrobial proteins of 18 kDa (CAP18), suggesting the 11-kDa polypeptide might be a homologue of CAP18. In contrast, the amino acid analysis of the 5-kDa antibacterial polypeptide revealed that the polypeptide was composed of 41 amino acids (5007 Da) containing 7 lysine, 10 arginine, and 2 cystine residues. However, sequence analysis indicated that the N-terminus of the 5-kDa polypeptide was likely blocked. The 11- and 5-kDa polypeptides showed almost the same antibacterial activities; ED50values were 30–35 nMagainstEscherichia coliand 90–120 nMagainstStaphylococcus aureus,which were 4- to 20-fold lower than those of defensins. Furthermore, the 11- and 5-kDa polypeptide retained the antibacterial activities even at the physiological concentration of NaCl (0.15M), although the antibacterial activity of defensin was completely lost in the presence of NaCl.

Requirement of ADP-ribosylation for the pertussis toxin-induced alteration in electrophoretic mobility of G-proteins

Pertussis toxin (PTX) catalyzes the ADP-ribosylation of the alpha-subunit of GTP-binding proteins (G-proteins) in the presence of NAD+. Pertussis toxin also decreases the electrophoretic mobility of the alpha-subunit on urea SDS PAGE. This effect of PTX has been suggested to be a property of the toxin different from its ability to catalyze ADP-ribosylation. However, the present report provides evidence to the contrary; ie, this mobility shift required the ADP-ribosylation of alpha-subunits. This conclusion was based on: (1) in the presence of increasing concentrations of NAD+ (0.026-1.3 microM), there was a linear increase in the formation of the slower migrating alpha-subunit as measured by immunoblotting with selective antisera, (2) addition of NADase to the incubation mixture completely eliminated the formation of this protein, and (3) increasing concentrations of nicotinamide (50-250 mM), which inhibits ADP-ribosylation, decreased the amount of the slower migrating alpha-subunit. Thus, in addition to PTX, NAD+ was required for the mobility shift and the slower migrating alpha-subunit is likely the ADP-ribosylated form.

Purification and properties of an extracellular xylanase from the thermophilic fungus Humicola grisea var. thermoidea

Humicola grisea var. thermoidea mycelium grown on xylan as the sole source of carbon produced at least two extracellular xylanases. The main xylanolytic component (form 2; 90% of recovered activity) was purified to homogeneity. The apparent molecular mass of the purified enzyme was estimated to be 23 000 and 25 550 Da by Bio-Gel P-60 filtration and urea–SDS–PAGE, respectively. The purified enzyme was a glycoprotein with 45% carbohydrate content and pH and temperature optima of 5.5 and 70 °C, respectively. The apparent Km and Vmax values determined with larch-wood xylan were 3.3 mg/mL and 229 μmol∙min−1∙mg protein−1, respectively. The enzyme was highly specific for xylan and degraded this substrate to produce xylo-oligosaccharides, suggesting that it is a β-1,4-endoxylanase (EC 3.2.1.8). The minor enzymatic component of H. grisea extracellular xylanase activity (form 1) was partially purified and some of its properties were studied for comparative purposes. The results obtained suggested that the mode of action of xylanases form 1 and 2 on xylan differs. Key words: xylanase, hemicellulase, enzyme purification, endoxylanase, Humicola grisea.

A synthetic protein corresponding to the entire vpr gene product from the human immunodeficiency virus HIV‐1 is recognized by antibodies from HIV‐infected patients

The 95 amino acid-protein encoded by the non-structural vpr gene of the human immunodeficiency virus type 1 (LAV-1BRU isolate) was chemically synthesized by solid phase methodology. The synthetic vpr protein was characterized by amino acid analysis, sequence analysis, RP-HPLC, and urea-SDS PAGE. Using a radioimmunoassay, antibodies to the synthetic protein were detected in sera of 25% of HIV 1-seropositive patients tested. Western blot analysis suggested that the antibodies preferentially recognize the dimeric form of vpr.