Balanced protein samples (30 μg per lane) were heated for 5 min at 95°C and then loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the gels were transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore Corporation, Billerica, MA, USA) at 4°C by the wet transfer method (200 mA, 3 h). Following several rinses with phosphate buffer solution-Tween (PBST, containing 0.1% Tween-20), the transferred PVDF membrane was blocked with 5% nonfat milk in PBST for 1 h at room temperature. The membrane was then incubated for 3 h at room temperature with a rabbit polyclonal antibody against CRMP2 (Cell Signaling Technology, Danvers, MA, USA) and a mouse monoclonal antibody against αII-spectrin (Merck Millipore, Darmstadt, Germany). A mouse monoclonal antibody against β-actin (Santa Cruz Biotechnology, Paso Robles, CA, USA) was employed to verify equal loading of the samples. Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology, Paso Robles, CA, USA) was used as the secondary antibody. After incubation with the primary and secondary antibodies, a chemiluminescent detection kit (Immobilon-P, Millipore Corporation, Billerica, MA, USA) was used to visualize the protein bands. The relative band intensities were tested by densitometry using FluorChem FC2 software (ProteinSimple, CA, USA).
Immobilon p
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, Ireland, Morocco, Switzerland, Sweden, France, Italy, Spain, China, Canada, Israel, Denmark
Immobilon-P is a polyvinylidene fluoride (PVDF) membrane designed for use in Western blotting applications. It provides a high-binding capacity for proteins and offers good mechanical strength and low background. The membrane is chemically stable and has a pore size of 0.45 μm.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (45 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (42 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (38 mentions)
Protease inhibitor cocktail, by Roche (34 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (33 mentions)
Clarity western ecl substrate, by Bio-Rad (28 mentions)
Complete protease inhibitor cocktail, by Roche (26 mentions)
Protein assay, by Bio-Rad (26 mentions)
β actin, by Merck Group (21 mentions)
Supersignal west pico, by Thermo Fisher Scientific (21 mentions)
Odyssey infrared imaging system, by LI COR (20 mentions)
Image lab software, by Bio-Rad (20 mentions)
Anti β actin, by Merck Group (20 mentions)
Ecl prime, by GE Healthcare (20 mentions)
Ecl western blotting detection reagent, by GE Healthcare (20 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (19 mentions)
Bradford assay, by Bio-Rad (18 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (17 mentions)
Chemidoc mp imaging system, by Bio-Rad (17 mentions)
Protease inhibitor, by Roche (17 mentions)
1 644 protocols using immobilon p
1
Western Blot Analysis of CRMP2 and α-Spectrin
2
Western Blot Protocol for O-GlcNAc Detection
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Protein concentrations were determined, and lysates were reduced in 6X sample loading buffer (0.5 M Tris-HCl PH 6.8, 10% SDS, 30% glycerol, 0.2% 2-mercaptoethanol, 0.012% bromophenol blue), boiled for 5 min, separated by SDS PAGE (15 µg of protein/lane), and transferred to Immobilon-P (Millipore) PVDF membrane. Immunoblotting was performed using a rapid immunodetection method for Immobilon-P (Millipore Technical Note TN051). Briefly, the membranes were equilibrated in methanol and air-dried. The dry membrane was incubated in anti-O-GlcNAc antibody CTD110.6 in 1% casein/phosphate-buffered saline (PBS) overnight at 4°C and then washed three times in PBS, as previously described (Zou et al., 2012 (link)). Other membranes were equilibrated in PBS; incubated in 5% milk with Tris-buffered saline with 0.01% Tween 20 (TBST) for 1 h for blocking; washed three times in TBST and then incubated with appropriate antibodies in 5% milk/TBST overnight at 4°C and then washed three times in TBST. The membrane was incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After further washing in PBS or TBST the immunoblots were developed with enhanced chemiluminescence (PerkinElmer Life Sciences) using either using either autoradiograph film or digitally using the Amersham Imager 600.
3
Pancreas Lysate Preparation Protocol
4
Co-Immunoprecipitation of FLS2 and CRKs
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For Co-IP assays, the plasmids of Pro35S: FLS2-HA (modified pEarleyGate100 with a AvrII-3xHA-SpeI fragment introduced after the attR2 recombination site) and Pro35S: CRKs-GFP, Pro35S: FLS2-HA and Pro35S: GFP, or Pro35S: FLS2-HA and Pro35S: RBI2B-GFP were transformed into Arabidopsis protoplasts by polyethylene glycol (Sigma) for transient expression (Yoo et al., 2007 (link)). Total proteins were extracted with 0.5 mL protein extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 10 mM DTT, 10 mM EDTA, 1 mM NaF, 1 mM Na2MoO4·2H2O, 1% [w/v] polyvinylpyrrolidone, 1% [v/v] IGEPAL CA-630 [Sigma-Aldrich] and 1% [v/v] Roche protease inhibitor cocktail) and incubated with gentle shaking at 4°C for 1 h. Samples were then centrifuged at 14000 rpm for 15 min at 4°C. Proteins were separated by SDS–PAGE and then transferred to a polyvinylidine fluoride membrane (Immobilon-P; Millipore). Supernatants (1.5 mL) were adjusted to 2 mg/mL protein and incubated for 2 h at 4°C with 20 mL GFP Trap-A beads (Chromotek). Following incubation, beads were washed four times with TBS containing 0.5% (v/v) IGEPALCA-630. Proteins were separated by 8% SDS–PAGE and then transferred to a polyvinylidine fluoride membrane (Immobilon-P; Millipore). GFP and HA fusion proteins were detected by immunoblotting with anti-GFP and anti-HA primary antibodies, respectively.
5
Osteogenic Protein Expression Analysis
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Immunoblotting was done to check the expression levels of osteogenic proteins as well components that are involved in the Wnt/beta catenin pathway. For this purpose, cultured MCO cells were transfected with different oligo miRNAs and si-RNAs and incubated for 48h in the differentiation medium. Whole cell lysate was extracted with mammalian cell lysis buffer comprising protease inhibitor cocktail and phosphatase inhibitor (Sigma, St. Louis, MO, USA). Protein concentration was estimated by BCA assay, and then separated on different percentage of SDS-PAGE, which were then electroblotted onto PVDF membranes (Immobilon-P, Millipore, Billerica, MA, USA). The membrane was then probed with Runx-2, Type I collagen, Akap-3, Wnt-3a, LRP-6, β-Catenin, P-β-catenin, Lef-1 and β-actin antibodies as primary antibodies and incubated with secondary antibodies conjugated with Horseradish Peroxide (HRP) at 4°C overnight. Antibody details were given in Supplementary Table 2
6
Immunoblotting Assay for Whole Cell Lysates
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Immunoblotting assays for whole cell lysates of cells and tissues were performed as previously described (Tarrago et al., 2018 (link)). Tissues or cells were homogenized and lysed in NETN buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40) supplemented with 50 mM β-glycerophosphate, 5 mM NaF and a protease inhibitor cocktail (Roche). After 30 min of incubation at 4°C, the samples were centrifuged at 12,000 r.p.m. for 10 min at 4°C. Protein concentrations in the supernatants were determined by Bio-Rad protein assay. Lysates were separated by SDS–PAGE, and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore). Enhanced chemiluminescence detection was performed using Super-Signal West Pico or Femto Chemiluminescence Substrate (Thermo Scientific). Films were scanned and densitometry was performed using ImageJ. The following antibodies and their dilutions were used for immunoblotting: mouse CD38 (R&D Systems; AF4947, 1:1,000), CD45 (Abcam; AB40763; 1:1000), Phospholamban (PLN) (Invitrogen; MA3-922; 1:1000), CYP3a4 (Proteintech, 18227-1-AP; 1:1000), actin (Cell Signaling Technology; 8457, 1:5,000) and GAPDH (Cell Signaling Technology; 97166, 1:5,000).
7
Quantitative Proteomic Analysis of Alzheimer's Disease
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Postmortem MFG tissues were homogenized in TBS buffer at a ratio of 1:10 (wt/vol) with Pierce Protease and Phosphatase Inhibitor Cocktail (A32965, ThermoScientific) on ice. Tissue lysate was sonicated and then centrifuged at maximum speed for 15 min at 4 °C. Protein concentrations were measured using the BCA protein assay kit (Bio-Rad Laboratories, Inc.). Electrophoresis was performed using 30 μg of protein lysates, resolved in a 4–12% SDS-PAGE gel (CriterionTM TGXTM, Bio-Rad Laboratories, Inc.) and transferred to a nitrocellulose membrane (Immobilon®-P, Millipore) that was blocked with 5% BSA in TBS with 0.01% tween, followed by overnight incubation of primary antibodies; HT7 (1:300, MN1000, Thermo Fisher), AT8 (1:1000, MN1020, Invitrogen) and PHF1 (1:1000, Peter Davies antibodies) diluted in the blocking solution. Horseradish peroxidase (HRP) secondary antibodies (goat anti-mouse HRP conjugated (1:10,000, 626820, Invitrogen) were incubated for 2 h at RT and the proteins were detected with Supersignal West Pico (34580, Thermo Scientific) and imaged by using iBright 1500 (Invitrogen). Western blots were analyzed using ImageJ Fiji.
8
Western Blotting and Immunoprecipitation Methods
9
MFHR1 Protein Concentration Determination
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The concentration of mossMFHR1 used for activity assays was measured via ELISA, using the same antibodies and protocol performed for mossFH as described above. As standard for the ELISA, a batch of purified mossMFHR1 was used in which its concentration was determined via band densitometry (Quantity One, Bio-Rad, Munich, Germany) after SDS–PAGE (Ready Gel Tris-HCl, Bio-Rad) and Coomassie staining. The sandwich ELISA using this MFHR1 standard protein has a linear response between 1 and 64 ng/ml. Electrophoretic separation of proteins was carried out in 10% SDS–polyacrylamide gels (Ready Gel Tris-HCl; BioRad) at 120 V. Subsequently, gels were stained with PageBlue® Protein Staining Solution (PageBlue™, Thermo Fisher Scientific). For Western blot analysis, SDS-PAGE gel was blotted to polyvinylidene fluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA, USA) in a Trans-Blot SD Semi-Dry Electrophoretic Cell (Bio-Rad) for 1 h with 1 mA /cm2 membrane. Immunoblotting was performed using mouse anti-His antibodies (MAB050, R&D Systems, Minneapolis, MN, USA) as primary and sheep anti-mouse antibodies coupled to peroxidase (NA931, Amersham ECL™, GE Healthcare) as secondary antibody in a 1:500 and 1:10,000 dilution respectively, followed by chemiluminescence development (ECL™ Advance Western Blotting Detection Kit, GE Healthcare) following the manufacturer’s instructions.
10
Subcellular Protein Fractionation and Western Blotting
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Cells were lysed in the SDS/Nonidet P-40 lysis buffer [1% SDS, 1% Nonidet P-40, 50 mM Tris (pH 8.0), 150 mM NaCl, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM NaF, and 100 µM Na3VO4]. Nucleus and cytosol protein fractions were extracted using Nuclear/Cytosol Fractionation kit (BioVision). The lysates were boiled for 5 min and then cleared by centrifugation at 15,000 rpm and 4°C. A Bradford protein assay reagent (BioRad) was used to determine the protein concentration of supernatants. Lysates were further boiled for 5 min in the sample buffer. Then, samples were resolved using the SDS-PAGE and transferred onto Immobilon-P (Millipore Corp.) sheets. Blots were first incubated in blocking buffer [5% (w/v) nonfat dry milk in Tris-buffered saline (TBS) plus 0.05% Tween 20] for 30 min. Then, they were incubated with a primary antibody for 16 h at 4°C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 30 min at room temperature (RT). The ECL-plus chemiluminescence (GE Healthcare) was used to visualize antibody-antigen complex.
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