2-de rehydration buffer  (Bio-Rad)

Journal: Pathogens

Article Title: Trichomonas vaginalis Legumain-2, TvLEGU-2, Is an Immunogenic Cysteine Peptidase Expressed during Trichomonal Infection

doi: 10.3390/pathogens13020119

Figure Lengend Snippet: Effect of glucose on the mRNA and protein expression of TvLEGU-2. ( A ) qPCR with specific primers for the tvlegu-2 and α-tubulin genes using 100 ng of cDNA from parasites grown in glucose restriction (GR) and high-glucose (HG) conditions. α-tubulin was used as a normalizing gene; ns, no significant differences. ( B ) Western blot assay of PREs from parasites grown under GR (<1 mM) and HG (50 mM). Coomassie Brilliant-Blue (CBB)-stained 10% SDS PAGE gel for PREs from parasites grown under GR and HG conditions (Lanes 2 and 3), respectively. For WB assays, duplicate gels were transferred onto NC membranes and incubated with different antibodies: Rα-TvLEGU-2pep (1:500 dilution) to detect the TvLEGU-2 protein, Rα-TvCP2r (1:6000 dilution) to detect a control protein overexpressed under GR conditions, Rα-TvTIM (1:1000 dilution) to detect an overexpressed TvTIM control protein under HG conditions, and a negative control with PI serum or no primary antibody (−). Arrowheads show the position of the native TvLEGU-2 (∼55, ∼49, ∼34, and ∼29 kDa) protein bands. ( C ) Silver-stained 2-DE protease-rich extracts from parasites grown in normal glucose conditions (25 mM) ( Ca ). WB of duplicate gels transferred onto NC membranes incubated with Rα-TvLEGU-1r (1:1000 dilution) antibody ( Cc ) to serve as a control for the specificity of Rα-TvLEGU-2pep antibody against other legumain proteins, Rα-TvLEGU-2pep (1:100 dilution) ( Cd ) to detect the TvLEGU-2 protein in PREs, or a negative control with PI serum or no primary antibody (−) ( Cb ). Arrowheads show the position of the native TvLEGU-2 (∼34 and ∼29 kDa) proteins. ( D ) Coomassie Brilliant Blue-stained, purified recombinant TvLEGU-1r protein (CBB; Lane 1). WB assays of TvLEGU-1r incubated with Rα-TvLEGU-1r (1:3000 dilution) (Lane 3), or Rα-TvLEGU-2pep (1:1000 dilution) (Lane 4) antibody or PI serum or only the secondary antibody as a negative control (−) (Lane 2). Arrowhead points to the recombinant protein band TvLEGU-1r (∼46 kDa). ( E ) Coomassie Brilliant Blue-stained, purified recombinant TvLEGU-2r protein (CBB; Lane 1). WB assays of TvLEGU-2r incubated with Rα-TvLEGU-2pep (1:1000 dilution) (Lane 4), or Rα-TvLEGU-1r (1:3000 dilution) (Lane 3) antibody, or PI serum or only the secondary antibody as a negative control (−) (Lane 2). Arrowhead points to the recombinant protein band TvLEGU-2r (∼85 kDa). kDa, molecular weight markers in kilodaltons (Bio-Rad).

Article Snippet: For 2-DE assays, PRE was obtained from parasites (6 × 10 7 ) lysed directly in 2-DE rehydration buffer (Bio-Rad, Hercules, CA, USA) in the absence of proteinase inhibitors.

Techniques: Expressing, Western Blot, Staining, SDS Page, Incubation, Negative Control, Purification, Recombinant, Molecular Weight

Journal: Pharmaceutical Biology

Article Title: Differential proteomic analysis of platelets suggested target-related proteins in rabbit platelets treated with Rhizoma Corydalis

doi: 10.1080/13880209.2016.1229340

Figure Lengend Snippet: The 2-DE proteome images of control (A) and RC-treated (B) platelets. The differentially expressed protein spots were shown by the arrows.

Article Snippet: Two-dimensional electrophoresis analysis was performed on a GE 2-DE system.

Techniques:

Journal: Plant physiology

Article Title: Voltage-dependent anion channel proteins associate with dynamic Bamboo mosaic virus-induced complexes.

doi: 10.1093/plphys/kiab519

Figure Lengend Snippet: Figure 1 Identification of the BaMV-induced proteins in the S100 fraction. A, Genome of BaMV. B, Schematic representations of infectious con- structs which were BaMV-derived in this study. pKn, a binary vector which can express foreign genes driven by the upstream double 35S promoter of cauliflower mosaic virus. It also has a nopaline synthase (Nos) terminator. pKB, which has an infectious full-length BaMV construct in the pKn vector. pKB GFP, has a GFP ORF downstream of TGBp3, which was driven by the same promoter as CP. pKB GFP-2a-CP, GFP, and CP are linked with a 2a protein. C, 2-DE analysis of S100 fractions from vector- or BaMV-inoculated N. benthamiana. S100 fractions were extracted at 7 dpi. The proteins from S100 were separated by 2-DE analysis and the excised differentiated dots (indicated as numbers) were analyzed by LC–MS/MS. Agrobacterium tumefaciens bearing pKn or pKB plasmid was infiltrated into 28-d-old N. benthamiana. The infiltrated leaves were collected at 7 dpi. D, Total RNA was extracted from infiltrated leaves and mRNA levels of NbVDACs were quantified by RT-qPCR. Data are mean ± standard de- viation (SD) from at least three independent experiments with three replicates each (N 5 3, n = 3) Asterisks indicate statistically significant differ- ences between the indicated groups by Student’s t test (**P 5 0.01, ***P 5 0.001).

Article Snippet: The pellet was washed 3 times with methanol and resolved in 2-DE rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% IPG-buffer and 60 mM DTT) and separated by 13 cm Immobiline DryStrip pH 3–10 (GE healthcare, Chicago, IL, USA) with the conditions 500 V for 1 h, 1,000 V for 1 h, 2,000 V for 1 h, 8,000 V for 2.5 h with gradient and 8,000 V for 2 h. The strip was soaked in equilibration buffer I (50 mM Tris–HCl pH 8.8, 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate (SDS) and 1% dithiothreitol) for 15 min and transferred to equilibration buffer II (50 mM Tris–HCl pH 8.8, 6M urea, 30% glycerol, 2% SDS, and 135 mM iodoacetamide) for the other 15 min. Next, the strips were placed onto 10–16% gradient SDS–polyacrylamide gels sealed by a 1% low-melting point agarose solution containing 1% agarose, 25 mM Tris base, 192 mM glycine, 0.1% SDS, and 0.002% (wt/vol) bromophenol blue.

Techniques: Derivative Assay, Plasmid Preparation, Virus, Construct, Liquid Chromatography with Mass Spectroscopy, Quantitative RT-PCR