Total lysates were obtained using-modified RIPA lysis buffer as described previously [41 (link),42 (link),43 (link)]. The proteins were separated by SDS-PAGE and transferred onto an Immobilon-P membrane (GE Healthcare Life Science, Pittsburgh, PO, USA). The membranes were incubated with 5% nonfat milk for 30 min and then overnight with the primary antibodies. After overnight incubation, the membranes were incubated with secondary antibodies for 2 h, and were exposed using an enhanced chemiluminescence Western blot kit (EMD Millipore, Darmstadt, Germany). All protein band intensity was measured using ImageJ. For apoptosis analysis, cells were fixed with 100% ethanol for 2 h at 4 °C. Next, cells were incubated with RNase for 30 min at 37 °C and stained with propidium iodide. DNA content was measured by flow cytometry (BD Biosciences).
Immobilon p membrane
Manufactured by GE Healthcare
Sourced in United States
Immobilon-P membrane is a polyvinylidene fluoride (PVDF) membrane used for protein transfer and immobilization in Western blotting and other protein analysis techniques. It provides high protein-binding capacity and is compatible with a variety of detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence western blot kit, by Merck Group (3 mentions)
Ecl detection kit, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Alkaline phosphatase conjugated goat anti rabbit antibody, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Rabbit anti vcam 1, by Cell Signaling Technology (1 mentions)
Peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Ecl chemiluminescent detection system, by GE Healthcare (1 mentions)
Nupage novex, by Thermo Fisher Scientific (1 mentions)
Cellytic nuclear extraction kit, by Merck Group (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Blocking reagent, by Toyobo (1 mentions)
Can get signal solution 1, by Toyobo (1 mentions)
Ecl detection kit, by GE Healthcare (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
11 protocols using immobilon p membrane
1
Western Blot and Apoptosis Analysis
2
Immunoblotting of Membrane Proteins
3
Western Blot and Apoptosis Analysis
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Total lysates were obtained using modified RIPA lysis buffer as described previously [41 (link),42 (link),43 (link)]. The proteins were separated by SDS-PAGE and transferred onto an Immobilon-P membrane (GE Healthcare Life Science, Pittsburgh, PO, USA). The membranes were exposed using an enhanced chemiluminescence Western blot kit (EMD Millipore, Darmstadt, Germany). For apoptosis analysis, cells were harvested and fixed with 100 % ethanol for 2 h at 4 °C. Then cells were resuspended in 50 μg/mL RNase for 30 min at 37 °C, and added to 50 μg/mL propidium podide. The stained cells were analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA).
4
Protein Extraction and Western Blot Analysis
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Cells were washed with cold PBS and lysed on ice in 50 µL of lysis buffer (50 mM Tris-HCl, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, pH 7.5) [53 (link), 54 (link)]. Lysates were centrifuged at 10,000 × g for 15 min at 4°C, and the supernatant fractions were collected. Proteins were separated by SDS-PAGE and transferred to an Immobilon-P membrane (GE Healthcare Life Science, Pittsburgh, PO, USA). Specific proteins were detected using an enhanced chemiluminescence (ECL) western blot kit (EMD Millipore, Darmstadt, Germany) according to the manufacturer’s instructions.
5
Western Blot Protein Analysis
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Cells were washed with cold PBS and lysed on ice in 50 μL of lysis buffer (50 mM Tris-HCl, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, pH 7.5). Lysates were centrifuged at 10,000 × g for 15 min at 4 °C, and the supernatant fractions were collected. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to an Immobilon-P membrane (GE Healthcare Life Science, Pittsburgh, PO, USA). Specific proteins were detected using an enhanced chemiluminescence Western blot kit (EMD Millipore, Darmstadt, Germany), according to the manufacturer’s instructions.
6
Apoptosis and Protein Expression Analysis
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For apoptosis analysis, cells were fixed with 100% ethanol for 2 hours at
4°C, resuspended in 250 μL of 1.12% sodium citrate buffer (pH 8.4)
containing RNase (12.5 μg/mL), and incubated for 30 minutes at 37°C.
After that, cellular DNA was stained with 250 μL of propidium iodide (50
μg/mL). The stained cells were analyzed by flow cytometer (BD Biosciences).
To examine protein expression, cells were lysed in RIPA lysis buffer and
centrifuged at 12,000 ×g at 4°C for 15 minutes. The
proteins were separated by SDS-PAGE electrophoresis and transferred to an
Immobilon-P membrane (GE Healthcare Life Science, Pittsburgh, PO, USA). The
specific membranes were detected using an Immobilon Western Chemiluminescent HRP
Substrate (EMD Millipore, Darmstadt, Germany).
4°C, resuspended in 250 μL of 1.12% sodium citrate buffer (pH 8.4)
containing RNase (12.5 μg/mL), and incubated for 30 minutes at 37°C.
After that, cellular DNA was stained with 250 μL of propidium iodide (50
μg/mL). The stained cells were analyzed by flow cytometer (BD Biosciences).
To examine protein expression, cells were lysed in RIPA lysis buffer and
centrifuged at 12,000 ×g at 4°C for 15 minutes. The
proteins were separated by SDS-PAGE electrophoresis and transferred to an
Immobilon-P membrane (GE Healthcare Life Science, Pittsburgh, PO, USA). The
specific membranes were detected using an Immobilon Western Chemiluminescent HRP
Substrate (EMD Millipore, Darmstadt, Germany).
7
Curcumin Modulates TNF-α-Induced Inflammatory Signaling
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HUVECs pre-exposed to curcumin or vehicle were harvested at the end of a 4-hour-TNF- activation period and washed twice with ice-cold PBS. Total proteins were extracted using lysis buffer containing 50 mmol/L Tris pH 7.8, 150 mmol/L NaCl, 0.5% sodium deoxycholate, 1% NP40, antiprotease and anti-phosphatase. Protein concentration was determined using BCA protein assay reagent Kit (Interchem). Lysates were loaded onto a 10% SDS-polyacrylamide gel for electrophoresis and then transferred onto immobilon-P membrane (GE Healthcare). The membrane was incubated in 5% (wt/vol) dried milk protein in TBS containing 0.05% Tween-20 for 1 hour, and then further reacted with primary antibodies: rabbit anti-iCAM (Santa Cruz, 1:1000), rabbit anti-vCAM-1 (1:1000, GTX), rabbit anti-NFB total (1:1000, Cell Signaling), rabbit anti-NFB phosphor-ser536 (1:1000, Cell Signaling), rabbit anti-IB total (1:1000, Cell Signaling) and rabbit anti-IB phosphor-ser32
(1:1000, Cell Signaling). After extensive washes, membrane was incubated with anti-rabbit IgG antibody conjugated to HRP (1:5000, Santa Cruz, USA). Protein bands were visualized using ECL detection kit (Millipore, USA) and then analyzed using Image J software (www.imagej.nih.gov).
Graphs represent protein level expressed as the mean +/-standard deviation of 4 independent experiments.
(1:1000, Cell Signaling). After extensive washes, membrane was incubated with anti-rabbit IgG antibody conjugated to HRP (1:5000, Santa Cruz, USA). Protein bands were visualized using ECL detection kit (Millipore, USA) and then analyzed using Image J software (www.imagej.nih.gov).
Graphs represent protein level expressed as the mean +/-standard deviation of 4 independent experiments.
8
Quantification of ALDH1 Protein by Western Blot
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Protein samples (30 μg) were separated on SDS-polyacrylamide gels by electrophoresis and the proteins were transferred to immobilon-P membranes (GE Healthcare). Level of the ALDH1 protein was determined using anti-ALDH1A1 antibodies (Abcam) at a concentration of 1 : 1000. Anti-enolase antibody (Santa Cruz Biotechnology, Santa Cruz) was used as housekeeping marker at a concentration of 1 : 1000. The bound antibodies were visualized with horseradish peroxidase- (HRP-) coupled secondary antibody and chemiluminiscence detection system.
9
Cytoplasmic Fractionation and Western Blot Analysis
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Cytoplasmic fractions were prepared from brain cortex and liver from 7-month-old male and female mice using the CelLytic NuCLEAR Extraction Kit (Sigma) following the manufacturer’s procedures. Protein extracts were aliquoted and stored at-80°C until used. Protein concentration was determined by using the Pierce BCA Protein Assay kit (Thermo Scientific). Between 50 to 150 μg of protein was run in denaturing 10% Bis-Tris SDS-polyacrylamide gels (NuPAGE Novex, Life Technologies) and transferred to Immobilon-P membranes (GE Healthcare, Piscataway, NJ). Membranes were blocked for 1 h in 5% low fat dried milk in TBS containing 0.1% Tween-20 (TBS-T) and then incubated for 1 h with the primary antibody. After washing in TBS-T, the membranes were incubated with peroxidase-conjugated secondary antibody (GE Healthcare) (1:5,000) for 1 h. Membranes were developed using the ECL chemiluminescent detection system (GE Healthcare). Equal protein load was confirmed after reprobing the membrane using anti-β-actin antibodies. The films were scanned and the densities of the bands measured using NIH ImageJ Software. The densities of the bands were normalized against those of β-actin and the mean ratios calculated. Statistical analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA).
10
Western Blot Analysis of Sperm β1 Integrin
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Capacitated WT or β1 KD sperm were washed twice in PBS, the pellet was snap-frozen in liquid N2 and stored at –80 °C for further use. Sperm aliquots (2 × 106) were lysed in 50 mM Tris (pH 8), 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate and 1% NP40, supplemented with Protease Inhibitor Cocktail (Sigma, St. Louis, MO, USA) for 1 h on ice, gently sonicated with an equal volume of 2x NuPAGE® LDS sample buffer (Thermo Fisher Scientific, Illkirch, France). Protein concentrations were determined by microBCA (Pierce, Thermo Fischer Scientific, Illkirch, France) and sperm proteins were separated by electrophoresis in Novex® Tris-glycine precast gel (Thermo Fisher Scientific, Illkirch, France) and electro-transferred to Immobilon-P membranes (GE Healthcare, France). Membranes were blocked for 1 h with 2% casein prior to incubation with primary anti-β1 integrin antibody at 1:250 for 90 min at 37°C, and anti-β-tubulin antibody (Merck-Millipore, Molsheim, France) at 1:15 000 for 90 min at RT followed by appropriate secondary HRP conjugated antibodies (0.2 µg/mL) at RT. HRP activity was revealed by ECL detection kit (Merck-Millipore, Molsheim, France) and ImageQuant LAS 4000 for quantitative imaging.
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