20(S)-PPD was prepared by chemically modifying ginseng saponins with periodic acid as described previously [12 ]. The 20(S)-PPD sample was dissolved in dimethyl sulfoxide (DMSO) at 10 mM as a stock solution and diluted to the indicated concentrations with phosphate-buffered saline (PBS) before use. Paclitaxel (Taxol™) was obtained from Bristol-Myers Squibb Pharmaceutical (Montreal, Quebec), dissolved in DMSO, and stored at −20°C in the dark. Antibodies against cleaved poly (ADP-ribose) polymerase (PARP) and caspase-9 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO). The annexin V-FITC/PI apoptosis detection kit was from BD Biosciences (San Diego, CA). The ECL membrane was from GE Healthcare Life Sciences (Pittsburgh, PA). RIPA buffer, the BSA protein assay kit, and the SuperSignal® West Pico Chemiluminescent Substrate kit were purchased from ThermoFisher Scientific (Massachusetts, MA). Fetal bovine serum (FBS) was obtained from Gibco-BRL (Carlsbad, CA). DMEM/F12 medium was from PAN Biotech (Aidenbach, Germany). All other chemicals, otherwise specified, were from Sigma-Aldrich (St. Louis, MO).
Ecl membrane
Manufactured by GE Healthcare
Sourced in France, Germany
ECL membranes are a type of laboratory equipment used in Western blotting, a common technique in molecular biology and biochemistry. These membranes serve as a substrate for the chemiluminescent detection of proteins after they have been separated and transferred from a gel. The core function of ECL membranes is to facilitate the visualization of specific proteins on a membrane through the use of chemiluminescent reagents.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2h tetrazolium bromide mtt, by Merck Group (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bsa protein assay kit, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by BD (1 mentions)
Dmem f12 medium, by PAN Biotech (1 mentions)
Antibodies against β actin, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Penta his antibody, by Qiagen (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Ampholyte, by GE Healthcare (1 mentions)
2 d clean up kit, by GE Healthcare (1 mentions)
Chaps, by Merck Group (1 mentions)
Thiourea, by Merck Group (1 mentions)
Grinding kit, by GE Healthcare (1 mentions)
7 protocols using ecl membrane
1
Synthesis and Characterization of 20(S)-PPD
2
Osteoblast HIF-1α Protein Analysis
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Following incubation at 2% O2, osteoblasts were removed from the incubator and immediately lysed in RIPA buffer containing a protease inhibitor cocktail. Whole cell lysates were then separated by SDS–polyacrylamide gel electrophoresis, blotted onto an enhanced chemiluminescence (ECL) membrane (GE Healthcare), probed with an HIF-1α polyclonal antibody at 1:500 dilution (Cell Signaling Technology), and visualized using ECL Plus detection (GE Healthcare).
3
Quantifying Parasite Protein Expression
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Total protein was recovered from 6.5x106 un-induced or induced parasites grown continuously until 72 h. Whole cell extracts were electrophoresed on an SDS-PAGE (12% gel) and transferred to ECL membrane (GE Healthcare); Anti-TbRRM1 mouse antiserum was used at 1:1000 dilution. Either anti-transaldolase (TAL) or anti-enolase (Enol) rabbit sera were used as loading control. Peroxidase-labeled goat anti-mouse or goat anti-rabbit (both from Thermo Scientific) were used as secondary antibodies. SuperSignal West Pico from Thermo Scientific was used as chemiluminescent substrate.
4
Western Blot Analysis of AGR2, FOXA2, and GAPDH
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Cells were lysed in RIPA buffer (Rockland Immunochemical Inc., Limerick, PA) supplemented with complete protease inhibitors (Roche, Nutley, NJ), and protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). Proteins (50 μg per lane) were separated on a 10% SDS-PAGE gel and then transferred onto ECL membranes (GE Healthcare, Piscataway, NJ). Membranes were incubated with primary antibodies against AGR2 (1/10,000 dilution; Abcam, Cambridge, MA), FOXA2 (1/2,000 dilution; Abcam), or GAPDH (1/2,000 dilution, Santa Cruz Biotech Inc., Texas, TX), and then with a HRP labeled anti-mouse IgG secondary antibody (Jackson ImmunoResearch Inc., West Grove, PA). The membranes were incubated with ECL substrates for 1 min, and target proteins were visualized with a ChemiDoc XRS Imaging System (Bio-Rad, Hercules, CA).
5
Western Blot Analysis of Protein Phosphorylation
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After treatment, cells were solubilized in lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 20 mM EDTA, 100 mM NaF, 10 mM Na3VO4, 1 mM PMSF, 1mM aprotinin and 0.5% NP40) and incubated for 30 min at 4°C. Lysates were centrifuged at 12,000 g for 15 min at 4°C and supernatants were adjusted with Laemmli SDS sample buffer. Proteins were separated by SDS/PAGE and transferred to ECL membranes (GE Healthcare, Velzy-Villacoublay, France) in a Tris (20 mM), glycine (150 mM), and ethanol (20%) buffer at 100 V for 1 h at 4°C. Antibodies were incubated in saturation buffer (50 mM Tris pH 7.5, 50 mM NaCl, 0.2% Tween, 5% BSA) and revealed with a secondary-peroxidase-conjugated antibody followed by ECL detection (GE Healthcare). Protein bands were quantified using the ImageJ software. The signal of a given total protein was normalized to the signal of Hsp60, used as a loading control from the same sample. Bands corresponding to phosphorylated proteins were normalized to the signal of the corresponding total protein which had been normalized to Hsp60.
6
SDS-PAGE and Immunoblotting for Recombinant Proteins
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Recombinant proteins were separated by SDS-PAGE and transferred onto ECL membranes (GE Healthcare, Freiburg, Germany). Protein gel blots were saturated with Penta-His antibody (Qiagen, Hilden, Germany) in a dilution of 1/2000. Goat anti-mouse antibody conjugated to horseradish peroxidase (HRP) (Pierce, Bonn, Germany) was used as secondary antibody in a dilution of 1/2000. Super Signal West Dura Extended Duration Substrate (Pierce, Bonn, Germany) was utilized for antigen detection. Blots were exposed to X-ray films.
7
Protein Extraction and 2D-Gel Separation
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Tissue homogenization and 1-DE protein separation were done as previously described (11) .
Briefly, 100 mg of each tissue was homogenized in a detergent solution (4% Triton X100, 1X antiprotease cocktail; Sigma, St Louis, MO, USA) and ground using a grinding kit (GE Healthcare) before protein precipitation with a 2D cleanup kit (GE Healthcare). The supernatant was removed and the pellet was resuspended in 250 ml of sample buffer (8 M urea/2 M thiourea [Sigma], 4% CHAPS [Sigma]). Protein concentration was determined using the Bradford assay (BioRad). Proteins (500 mg per gel) were eluted into rehydration buffer (8 M urea/2 M thiourea [Sigma], 2% CHAPS [Sigma],
DeStreak reagent [15 mg/ml, GE Healthcare] and ampholytes [1% IPG buffer, GE Healthcare]) before first separation according to their isoelectric points along a nonlinear immobilized pH-gradient (IPG) strip (pH 3-11 NL, 18 cm long) using an IPGphor III apparatus (GE Healthcare), as described elsewhere (14) . For the second dimension, equilibrated strips were loaded onto 8-18% SDSpolyacrylamide gels and electrophoresis was performed as in (19) . One preparative gel was stained with CBB G-250 (Sigma) and used for spot cutting and protein sequencing. The remaining gels were electroblotted onto ECL membranes (GE Healthcare).
Briefly, 100 mg of each tissue was homogenized in a detergent solution (4% Triton X100, 1X antiprotease cocktail; Sigma, St Louis, MO, USA) and ground using a grinding kit (GE Healthcare) before protein precipitation with a 2D cleanup kit (GE Healthcare). The supernatant was removed and the pellet was resuspended in 250 ml of sample buffer (8 M urea/2 M thiourea [Sigma], 4% CHAPS [Sigma]). Protein concentration was determined using the Bradford assay (BioRad). Proteins (500 mg per gel) were eluted into rehydration buffer (8 M urea/2 M thiourea [Sigma], 2% CHAPS [Sigma],
DeStreak reagent [15 mg/ml, GE Healthcare] and ampholytes [1% IPG buffer, GE Healthcare]) before first separation according to their isoelectric points along a nonlinear immobilized pH-gradient (IPG) strip (pH 3-11 NL, 18 cm long) using an IPGphor III apparatus (GE Healthcare), as described elsewhere (14) . For the second dimension, equilibrated strips were loaded onto 8-18% SDSpolyacrylamide gels and electrophoresis was performed as in (19) . One preparative gel was stained with CBB G-250 (Sigma) and used for spot cutting and protein sequencing. The remaining gels were electroblotted onto ECL membranes (GE Healthcare).
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