The molecular weight of the purified recombinant BpEG was determined using 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970 (link)) using the Opti-Protein Marker/Ladder (Applied Biological Materials, Inc., Canada). Protein bands were detected using Coomassie Brillant Blue (CBB) R-250 staining. The expression of BpEG protein samples was characterized using Western blotting with the standard protocols (Laemmli, 1970 (link); Towbin et al., 1979 (link)). The purified BpEG protein was also loaded on to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted for 1 h onto the polyvinylidene fluoride (PVDF) membrane in a Tris-glycine-methanol buffer. After, the membrane was incubated in a 1% bovine serum albumin (BSA) solution overnight and later probed with 6 × His-tag antibodies at 1:1,000 dilutions and a rabbit Anti-Mouse IgG (H&L) AP-conjugated with alkaline phosphatase was used as the secondary antibody at a dilution of 1:7,500 (Promega, United States). Chromogenic detection of BpEG protein bands was performed using western blue, a stabilized substrate for alkaline phosphatase (Promega, United States), and the reaction was later stopped by rinsing with water.
Western blue
Manufactured by Promega
Sourced in United States
Western Blue is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It provides a sensitive and stable signal for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Sds sample buffer, by Nacalai Tesque (1 mentions)
Anti lexa antibody, by Merck Group (1 mentions)
Biotinylated secondary antibody, by GE Healthcare (1 mentions)
Anti h3k27me3, by Cell Signaling Technology (1 mentions)
Anti pan histone h3, by Abcam (1 mentions)
Anti ezh2, by Cell Signaling Technology (1 mentions)
Mouse anti αsyn antibody, by BD (1 mentions)
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
Anti jag1, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Smz800 microscope, by Nikon (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Bca protein assay reagent, by Beyotime (1 mentions)
Chemianalysis image analysis software, by Clinx (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Cellytic m, by Merck Group (1 mentions)
Ac 74, by Merck Group (1 mentions)
17 protocols using western blue
1
Characterization of Recombinant BpEG Protein
2
Quantitative analysis of phosphorylated AQP2 in urine exosomes
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Urine exosome samples were denatured in SDS sample-buffer (Nacalai Tesque, Kyoto, Japan) for 20 min at 75 °C. Then, the samples were separated by SDS-PAGE and the proteins were transferred to a PVDF membrane (Immobilon-P, Merk KGaA, Darmstadt, Germany). The blots were probed with following primary antibodies: rabbit anti AQP2 antibody for total AQP2 [5 (link)], rabbit anti pS256-AQP2 antibody (Abcam, Cambridge, UK) [13 (link), 14 (link)], rabbit anti pS261-AQP2 antibody (PhosphoSolutions, Aurora, USA) [14 (link)], rabbit anti pS264-AQP2 antibody (PhosphoSolutions) [15 (link)] and rabbit anti pS269-AQP2 antibody (PhosphoSolutions) [14 (link)]. Alkaline-phosphatase-conjugated anti rabbit IgG antibody (Promega, Madison, USA) was used as a secondary antibody and Western blue (Promega) was used to detect the signals [13 (link)]. The band intensities of the western blots were quantified using ImageJ software (https://imagej.nih.gov/ij/ ).
3
Western Blot Analysis of Yeast Co-transformants
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The co-transformant yeasts were cultured in the vectors’ selective medium (–LW) overnight, diluted into the same medium, and cultured to mid-log phase at 30 °C for 7 h. Then, a cell amount corresponding to 4 OD600 was collected by centrifugation. The cell pellets were suspended in 30 µL of lysis buffer (80 mM Tris-HCl pH 6.8, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 2% sodium dodecyl sulfate (SDS), 100 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride (PMSF), 0.1 mg/mL bromophenol blue and yeast inhibitor proteases cocktail) and disrupted with glass beads after heating at 100 °C. Obtained cell lysates were supplemented with 70 µL of ESB buffer and heated at 100 °C for 2 more min. Clear supernatants from centrifugation were then subjected to SDS-PAGE. Proteins transferred onto polyvinylidene difluoride (PVDF) membrane were immunoblotted with anti-LexA antibody (1:2000, DNA-binding domain, Millipore, Billerica, MA, USA) or anti-Gal4 antibody (1:2000, activation domain, Sigma, Darmstadt, Germany). A secondary anti-rabbit IgG antibody conjugated to alkaline phosphatase (1:30,000, Sigma) was used and colorimetric revelation was performed with a NBT/BCIP mixture (Western blue, Promega) for 1 h.
4
Western Blot Protein Analysis Protocol
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The cultured cells were suspended in western lysis buffer (55 mM Tris-HCl (pH 6.8), 0.72 M 2-mercaptoethanol, 2% SDS, 0.005% bromophenol blue, and 8% glycerol) and boiled for 5 min. After centrifugation at 14,000 xg for 5 min, each supernatant was collected as a cell lysate. An EZQ Protein Quantitation Reagent (Life Technologies) was used to measure the protein concentration of each sample. Each protein sample (5 to 10 μg) was loaded onto a SDS-PAGE mini-gel; separated proteins were then blotted onto a polyvinylidene difluoride membrane (Merck Millipore). After sequential exposures to a primary antibody, a biotinylated secondary antibody (GE Healthcare), and a streptavidin-conjugated alkaline phosphatase (GE Healthcare), each membrane was developed with Western Blue (Promega). The primary antibodies were used as follows: anti-aromatase (CYP19 product), #SM2222PS, Acris; anti-β-actin, #A1978, Sigma; anti-pan-histone H3, #ab1791, Abcam; anti-H3K27me3, #9733, Cell Signaling; anti-EZH2, #5246, Cell Signaling.
5
Immuno-Dot Blot Analysis of Alpha-Synuclein
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Blotted aliquots of αSyn (10 × 2 μl) were dried on nitrocellulose before being blocked for 1 h in Tris‐buffered saline (TBS: 10 mm Tris; 150 mm NaCl; pH 7.5) + Tween20 (TBST) containing 2% dried skimmed milk (Marvel; Premier Foods, St Albans, UK). Primary antibodies were diluted 1:1000 and secondary antibodies were diluted 1:5000 in the blocking buffer. Mouse anti‐αSyn antibody (catalogue number 610786; BD Biosciences, Clontech, Palo Alto, CA, USA) was used to detect the presence of αSyn regardless of structure, the secondary for which was anti‐mouse IgG conjugated to alkaline phosphatase (Apase; Promega, Madison, WI, USA). Rabbit polyclonal A11 antibody (catalogue number AHB0052; Invitrogen, Carlsbad, CA, USA) was used to specifically detect oligomeric αSyn (Kayed et al. 2003 ), the secondary for which was anti‐rabbit IgG conjugated to APase (Promega). Before and after the addition of secondary antibodies, the paper was washed thoroughly in TBST to remove non‐specific binding. A final wash in TBS was carried out to remove Tween20 prior to the addition of Western Blue (Promega), a substrate for APase, that enabled visualization of the target protein.
6
Western Blot Analysis of Cellular Proteins
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Cell lysates were prepared as described previously.29 (link) Equal amounts of protein (30 μg/well) were separated on a discontinuous sodium dodecyl sulfate-10% polyacrylamide gel and blotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The primary antibodies used are listed in the Online Supplementary Methods. Antibody signals were visualized using Western blue (Promega, Madison, WI, USA).
7
Western Blot Analysis of Protein Expression
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Cells were collected with RIPA buffer supplemented with Proteinase Inhibitor Cocktail (P8340, Sigma) after two times washing with PBS. Lysates were cleared by centrifugation at 4°C for 15 min at 12,000 g, and protein concentrations were determined using BCA Protein Assay Kit (Pierce). Samples were subjected to SDS-PAGE, and transferred to a membrane. Primay antibodies were anti-Vimentin (Cell Signaling, #5741)58 (link), anti-KSP33 (link), anti-Jag1 (Cell Signaling, #2620)59 (link) and anti-β-actin (Sigma, A1978)60 (link). Alkaline phosphatase conjugated antibody (Promega) was used as a second antibody. Detection was carried out with Western blue (Promega).
8
Immunolocalization of PtFLA6 in Bent Stems
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Segments (0.5 cm) of the bended stems for two weeks were fixed overnight in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.5). Sections (10 µm) were cut out and blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. Then the sections were incubated with the PtFLA6 antibody (diluted at 1:50 with 0.1 M PBS containing 0.1% BSA) at room temperature for 1 h. After rinsed three times in PBS (5 min each), the samples were immersed in the alkaline phosphatase-conjugate secondary antibody (diluted at 1:100) for 1 h and then stained with 150–200 µl of Western Blue (Promega, USA). When blue color appeared, images were captured under bright field using the ECLIPSE 80i microscope (Nikon, Japan) or SMZ800 microscope (Nikon, Japan). Pre-immune serum was used as the negative control.
9
Dose-dependent Ni-smelting Fume Exposure Proteomics
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Cells were treated with different concentrations of Ni-smelting fumes for different times and then harvested at 70%-80% confluence. Cellular protein was extracted by lysis buffer and centrifuged at 12,000 rpm for 10 min. Concentrations of the protein extracts were measured using BCA protein assay reagent (Beyotime, China) and a microplate reader (Molecular Devices, CA, USA). Cellular protein was subjected to 8%-15% sodium dodecyl sulfate polyacrylamide gel, and the resolved proteins were electroblotted onto PVDF (polyvinylidene fluoride) membranes. The membranes were blocked with nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 10 min at 37°C and incubated with appropriate primary antibodies at 4°C overnight and horseradish peroxidase conjugated secondary antibody for 30 min. The protein blots were visualized by incubation with Western Blue (Promega, USA) to detect positive protein bands, and protein blots were analyzed using ChemiAnalysis image analysis software (Clinx Science Instruments, China). Equal loading of the samples was determined by quantitation of proteins and by reprobing membranes for β-actin.
10
Western Blot Analysis of Apoptosis Regulators
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Cells were dissolved on ice in 250 µL ice‐cold lysis buffer (CelLytic M; Sigma‐Aldrich) containing protease inhibitor (Sigma‐Aldrich) as described previously.19 , 21 Equal amounts of protein (50 µg/well) were separated on a discontinuous SDS‐10% polyacrylamide gel and blotted onto a nitrocellulose membrane (Bio‐Rad). Western blot analysis was carried out using the following primary Abs: G‐9 (Santa Cruz Biotechnology) for Fas; FD19 (Santa Cruz Biotechnology) and anti‐FADD rabbit Ab (Cell Signaling Technology) for FADD; 1C12 (Cell Signaling Technology) and anti‐caspase‐8 rabbit Ab (GeneTex) for procaspase‐8/FLICE and caspase‐8; 5D8 (Santa Cruz Biotechnology) for c‐FLIPL; anti‐c‐FLIPγ/δ rabbit Ab (Sigma‐Aldrich) for c‐FLIPS; D5J1E (Cell Signaling Technology) for both forms and the cleaved c‐FLIPL; 38 (BD Bioscience) for RIP1; C‐2 (Santa Cruz Biotechnology) for BCL2; E18 (Abcam) for BCL‐xL; Y37 (Abcam) for MCL1; 31A1067 (Santa Cruz Biotechnology) for procaspase‐3 and the cleaved forms; 194C1439 (Santa Cruz Biotechnology) for cleaved PARP1; and AC74 (Sigma‐Aldrich) for β‐actin. Antibody signals were visualized using Western Blue (Promega). We quantified the expression levels by comparison with each β‐actin level using ImageJ version 1.52k (://imagej.nih.gov/ij/ ).
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