In parallel, after the supernatant was collected, bacterial pellets were resuspended in FSB plus 20% DTT. Pellet samples were boiled for 15 min. At the time of loading, samples were normalized to the same number of cells. Cytosolic and secreted protein samples were run on a 12.5% SDS-PAGE gel and transferred to a blotting membrane (Immobilon-P) with a wet mini trans-blot cell (Bio-Rad). Blots were blocked for an hour in Tris-buffered saline with Tween 20 and 5% skim milk, and probed with the goat anti-YopE antibodies (Santa Cruz Biotechnology), rabbit anti-YopD (gift from Alison Davis and Joan Mecsas), rabbit anti-RpoA (gift from Melanie Marketon), rabbit anti-LcrF [35 (link)], rabbit anti-IscR [25 (link)], and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotech). Following visualization, quantification of the bands was performed with Image Lab software (Bio-Rad).
Immobilon p
Manufactured by Bio-Rad
Sourced in United States
Immobilon-P is a polyvinylidene difluoride (PVDF) membrane designed for protein transfer and immobilization during Western blotting and other protein analysis techniques. It provides a high-binding capacity for the transfer and immobilization of proteins from electrophoresis gels.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (5 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (5 mentions)
Wet mini trans blot cell, by Bio-Rad (3 mentions)
Ecl kit, by Cytiva (2 mentions)
Goat anti yope, by Santa Cruz Biotechnology (2 mentions)
Semi dry trans blot cell, by Bio-Rad (2 mentions)
Trans blot semi dry transfer cell, by Bio-Rad (1 mentions)
Dig glycan differentiation kit, by Roche (1 mentions)
Hrp labelled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Chemidoc touch apparatus, by Bio-Rad (1 mentions)
Stain free precast gel, by Bio-Rad (1 mentions)
Stain free system, by Bio-Rad (1 mentions)
Ecl plus kit, by GE Healthcare (1 mentions)
Anti phospho ampka thr172, by Cell Signaling Technology (1 mentions)
Anti ha, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Trans blot, by Bio-Rad (1 mentions)
Enhanced chemiluminescence western blotting detection kit, by Thermo Fisher Scientific (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
20 protocols using immobilon p
1
Western Blot Analysis of Yersinia Proteins
2
Quantifying Type III Secretion Proteins
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Supernatant samples were obtained as described above for the type III secretion assay. In parallel, after the supernatant was collected, the pellet was resuspended in the same volume of FSB plus 20% DTT as the corresponding supernatant sample. The pellet samples were then boiled for 15 min. Cytosolic and secreted protein samples were subjected to 12.5% SDS-PAGE and transferred to a blotting membrane (Immobilon-P) with a trans-Blot semidry transfer cell (Bio-Rad). Blots were blocked overnight in Tris-buffered saline with Tween 20 and 5% skim milk and probed with a YopE primary antibody and a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotech). Following visualization, densitometric quantification of the bands was performed with Image Lab software (Bio-Rad) with the first DMSO-treated WT Y. pseudotuberculosis YopE band set to 1.00.
3
Lectin Blot Analysis of Sialic Acid Linkages
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LNCaP and C4-2B cell lysates were made from 80 to 90% confluent LNCaP and C4-2B cultures, using 0.5 ml lysis buffer containing 1% (v/v) Triton X-100, 1% (v/v) Nonidet P40 and the following inhibitors: aprotinin (10 μg/ml), leupeptin (10 μg/ml), PMSF (1.72 mM), NaF (1 mM), NaVO3 (500 μM) and Na4P2O7 (500 μg/ml). Aliquots of lysates, containing 25 μg of protein, were boiled for 5 min in SDS/PAGE sample buffer containing 5% (v/v) 2-mercaptoethanol, electrophoresed on 4–20% gradient or 7.5% SDS/PAGE and transferred to PVDF membranes (Immobilon-P) (Bio-Rad Laboratories). Lectin blot analysis was performed using the DIG glycan Differentiation kit (Roche) following the manufacturer's protocol. Briefly, membranes were blocked for 30 min in blocking solution, washed in TBS and stained for α2,3- or α2,6-linked sialic acids by incubating the membranes with DIG-conjugated lectins MAA (1:200) and SNA (1:1000), respectively, in TBS containing 1 mM MgCl2, 1 mM CaCl2 and 1 mM MnCl2 (pH 7.5) for 1 h. After washing, the membranes were incubated for 1 h with anti-DIG antibody conjugated with ALP (1:1000 in TBS), which was followed by three wash steps and staining with NBT/BCIP (1:50 in 0.1 M Tris-HCl, 0.05 M MgCl2 and 0.1 M NaCl at pH 9.5).
4
Quantitative Western Blot Analysis of Muscle Proteins
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Western blot analysis was performed on muscle homogenates as previously described [16 (link)]. Briefly, after electrophoretic separation on a 4–20% gradient acrylamide gel (Stain-free precast gel, Biorad, France) and electrotransfer to Immobilon P (Biorad, France), the membrane was incubated with primary antibodies and then HRP-labelled secondary antibodies (Jackson ImmunoResearch Laboratories). Signal quantification was performed using a ChemiDoc Touch apparatus (Biorad, France) and the Image Lab software (Biorad). The amount of the chosen protein in each sample was corrected for differences in loading using either the amount of myosin, GAPDH or the total amount of proteins using the stain free system from Biorad, and normalized to the amount of the same protein present in the control, set to 100% as described previously [15 (link)]. Ten human controls (muscle biopsy from individuals non-affected by neuromuscular disease) of different age have been used, from 3.5 to 64 years. For each sample (mouse and human), 2–3 Western blots have been performed, and the value for each sample corresponds to the mean ± SEM of the different Western blots.
5
Western Blot Analysis of Phospho-AMPK in Yeast
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Yeast cells were grown for 48 h in YPD liquid medium and, then, inoculated in fresh YPD (initial O.D.600nm 0.25). Flasks were incubated under shaking (200 rpm) at 30°C and growth was followed till O.D.600nm ~1.0 (after 5 h). Culture samples were collected and total protein homogenates were prepared according to Yaffe and Schatz method [46 (link)] with minor modifications. NaOH and β-mercaptoethanol were added to a final concentration of 0.2 M and 1%, respectively. Tubes were incubated on ice for 2 minutes and, then, trichloroacetic acid was added to a final concentration of 6%. After incubation on ice for 10 minutes, tubes were centrifuged at maximum speed for 5 minutes. Supernatant was discarded and pellets were stored at -20°C until use or promptly resusspended in Laemlli sample buffer to obtain a 10-O.D. suspension. Proteins were separated by electrophoresis (SDS-PAGE, 10% SDS-polyacrylamide gel, 100 V, 2 hours) and electrotransferred to Immobilon-P for 20 min at 18 V in 25 mM Tris, 192 mM glycine and 10% methanol, using a trans-blot semi-dry cell (BioRad). Membranes were treated with anti-phospho-AMPKa Thr172 (Cell Signaling) or anti-HA (Sigma) antibodies. Blots were detected using the chemiluminescence ECL Plus kit (GE Healthcare).
6
Western Blot Protein Analysis
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Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Immobilon-P, Bio-Rad Laboratories, Richmond, CA, USA). Equal amounts of protein (20–50 µg) were loaded in each lane. Uniformity of sample loading and transfer integrity were verified by staining with Ponceau-S. After transfer, the PVDF membranes were blocked with nonfat milk and incubated overnight at 4 °C with primary antibodies followed by secondary antibodies conjugated to horseradish peroxidase (HRP). The levels of β-tubulin as a reference were detected using polyclonal rabbit anti-β-tubulin antibody, tubulin having already been widely used in similar experiments with microtubule poisons [18 (link),19 (link),20 (link)]. Detection of the immunoreaction was performed with an enhanced chemiluminescence (ECL) kit (Amersham BioSciences, Amersham, UK). Protein band densities were quantified using ImageJ software (NIH, Bethesda, MD, USA) after scanning the images with a ChemiDoc MP Imaging System (Bio-Rad).
7
Analyzing Protein Expression in VX2 Carcinomas
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The total proteins were extracted and concentrated for analysis of the primary culture cells from the VX2-induced carcinomas using the Bradford assay (Bio-Rad, Hercules, CA, USA); the same procedure was performed for the normal rabbit buccal tissues (used as the control). Equal levels of the protein were boiled prior to polyacrylamide gel electrophoresis; the proteins were transferred onto a polyvinylidene difluoride membrane (cat. no. IPVH 00010, Millipore Immobilon P) using Bio-Rad's Transblot. The membrane was then blocked, treated with primary antibodies (CD-44: cat. no. SC-7297; Bmi-1: cat. no. GTX114008) and γ-tubulin (1∶2 000; cat. no. T6557), followed by secondary antibody, and finally detected using an Amersham's ECL kit. The relative expression levels were measured and normalized to the expression level of the positive control.
8
Western Blot Analysis of Bacterial Stress Proteins
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Bacteria were cultured for 19 h at 37℃ with vigorous shaking as described above. The cells were collected by centrifugation and resuspended at 20 optical density at 600 nm (OD600) units per mL in 2× Laemmli loading buffer. After boiling for 10 min, the protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were semi-dry transferred to polyvinylidene fluoride membranes (Immobilon-P; Bio-Rad, USA). Primary antibodies specific to RpoS (Santa Cruz, USA), GadA (glutamic acid deoxycarboxylase A; MyBioSource, USA), and DnaK (heat shock 70 kDa protein; Enzo, USA) were used, and anti-mouse rabbit IgG coupled to peroxidase (Enzo) was used as a secondary antibody according to the manufacturers' instructions. Blotted membranes were visualized by exposure to X-ray films by using the Western Detection Kit System (Abclon, Korea).
9
Viral Infection Proteome Analysis in MDCK Cells
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The MDCK cell line was grown to approximately 90% confluency in a 6-well plate (1 d), and the cells were infected with H1N1 and incubated for an additional 1 h. After the medium was replaced with DMEM, samples were treated at different concentrations. After 24 h, the cells were washed with chilled PBS and lysed with EBC lysis buffer [1 mM EDTA, 0.5% NP-40, 50 mM NaF, 120 mM NaCl, and 50 mM Tris-HCl (pH 7.6)]. A protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA) was used to measure the protein concentrations. SDS-polyacrylamide electrophoresis was performed using a gel electrophoresis kit. The PVDF membranes (0.45 μ m Immobilon-P, Bio-Rad Laboratories, Inc.) to which the proteins were transferred were incubated with the antibodies overnight at 4°C and with the secondary antibodies at room temperature for 1 h in Tris-buffered saline. Finally, the samples were evaluated using an enhanced chemiluminescence Western blotting detection kit (Thermo Fisher Scientific).
10
Western Blot Protein Quantification
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Kidneys were removed, snap‐frozen in liquid nitrogen, and homogenized in chilled lysis buffer as described (McCormick et al., 2011 ). Half‐kidneys were homogenized and centrifuged at 3,500g for 15 min at 4°C. Total protein quantification was established using a colorimetric assay (Bio‐Rad DC Protein Assay) and 40 µg protein per sample was separated on a 4%–15% precast gel (Bio‐Rad Criterion Stain‐Free) before being transferred to a 0.45 µm PVDF membrane (Immobilon‐P) overnight at 150 mA at 4°C. The membrane was then blocked using nonfat milk protein in PBS with 0.1% (w/v) TWEEN20 for 1 hr at room temperature before being incubated overnight with primary antibody at 4°C. Antibody binding was detected using an HRP‐conjugated secondary antibody and visualized using Western Lightning Plus ECL. Prior to transfer, the gel was imaged using the PXi4 gel imaging system (Syngene). Total protein for each lane was measured using GeneTools software (Syngene). Membranes were imaged using the PXi4 gel imaging system and bands were quantified using GeneTools software. Bands were normalized to total beta‐actin protein.
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