BBMVs prepared using the Ca2+ precipitation method, cortical membrane, and whole homogenate were obtained from mouse kidneys and used for immunoblotting and immunoprecipitation analyses as described previously35 (link),45 (link),48 (link),49 (link). Protein samples were heated at 95 °C for 5 min in sample buffer in the presence of 2-mercaptoethanol and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred by electrophoresis to Immobilon-P polyvinylidene difluoride (Millipore, Billerica, MA, USA) and treated with diluted antibodies. Signals were detected using Immobilon Western (Millipore)35 (link),45 (link),48 (link),49 (link).
Immobilon p polyvinylidene difluoride
Manufactured by Merck Group
Sourced in United States
Immobilon-P polyvinylidene difluoride (PVDF) is a laboratory product manufactured by Merck Group. It is a microporous membrane material designed for use in various analytical techniques, such as Western blotting and protein transfer applications. The core function of Immobilon-P PVDF is to provide a stable and efficient medium for the immobilization and transfer of proteins from electrophoresis gels to a solid support for further analysis.
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Lab products found in correlation
Immobilon western, by Merck Group (6 mentions)
Ecl plus detection kit, by Cytiva (2 mentions)
Mouse monoclonal anti actin antibody, by Merck Group (2 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse igg, by Jackson ImmunoResearch (2 mentions)
Ecl prime, by GE Healthcare (2 mentions)
Horseradish peroxidase, by Agilent Technologies (1 mentions)
Acc 034, by Alomone (1 mentions)
Chemidoc touch system, by Bio-Rad (1 mentions)
Western lighting plus ecl kit, by PerkinElmer (1 mentions)
Precast gel, by Bio-Rad (1 mentions)
Mini protean 3 system, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Hyglo quick spray, by Thomas Scientific (1 mentions)
Protran, by GE Healthcare (1 mentions)
Tgx precast gel, by Bio-Rad (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Westernbright ecl, by Advansta (1 mentions)
Las4000 imager, by GE Healthcare (1 mentions)
Anti drp1 epr19274, by Abcam (1 mentions)
12 protocols using immobilon p polyvinylidene difluoride
1
Immunoblotting and Immunoprecipitation of Kidney BBMVs
2
TRPV4 Protein Expression in Rat Cerebral Myocytes
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The expression of TRPV4 channel protein in freshly dissociated FHH rat cerebral arterial myocytes was examined by Western blot analysis. Protein samples were prepared from freshly dissociated FHH rat cerebral arterial myocyte, and also from whole brains of FHH rat and the control Sprague Dawley rat that display normal myogenic cerebral autoregulation, by homogenization in lysis solution (Camiolo buffer, 75 mM potassium acetate, 300 mM NaCl, 10 mM EDTA, 100 mM l-arginine basic salt, and 0.25% Triton-X 100, protease inhibitor mix). Protein concentrations were determined using the Bradford assay [25 (link)]. Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a membrane (Immobilon-P polyvinylidene difluoride, Millipore) at 25°C for 2 h at 15 V. Following 2 h blocking (with 5% skim milk in Tris-buffered saline with Tween-20 (TBST), the membrane was then incubated with polyclonal TRPV4 primary antibodies (ACC-034, 1:500, Alomone Laboratories) overnight at 4°C, and then with horseradish peroxidase (HRP)-conjugated goat anti-rabbit polyclonal secondary antibody (DakoCytomation; 1:1,500) for 1 h at room temperature. An ECL Plus Detection kit (Amersham Biosciences) was used to visualize bands.
3
Western Blot Analysis of Na/Pi Transporters
4
SDS-PAGE Protein Analysis Protocol
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Protein samples were heated at 95°C for 5 min in sample buffer in the presence of 2‐mercaptoethanol and subjected to 8% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The separated proteins were transferred by electrophoresis to Immobilon‐P polyvinylidene difluoride (Millipore) and treated with diluted antibodies. Signals were detected using Immobilon Western (Millipore).
5
Immunoblotting Analysis of Pituitary and Kidney Samples
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The whole homogenate was obtained from mouse pituitary glands and kidneys and used for immunoblotting analyses as previously described.(9 (link),25 (link)) Protein samples were added to the sample buffer in the presence of 2-mercaptoethanol and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred by electrophoresis to Immobilon-P polyvinylidene difluoride (Millipore, Billerica, MA) and treated with diluted antibodies. Signals were detected using Immobilon Western (Millipore).
6
Western Blotting for Renal Sodium Transporters
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Protein samples were in denatured with 2-mercaptoethanol and subjected to 8 or 10% SDS-PAGE. The separated proteins were transferred by electrophoresis to Immobilon-P polyvinylidene difluoride (Millipore, Billerica, MA, USA) and then treated with the following diluted antibodies. Immunoblot analyses were performed using the following primary antibodies: affinity-purified anti-Npt2a (1:4,000) (34 (link)), anti-Npt2c (1:3,000) (41 (link)), and anti-Npt2b (1:2,000), as described previously (42 (link)). Anti-Klotho (for mouse total lysate; Trans Genic Inc., Fukuoka, Japan) was used following the manufacturer’s instructions. Mouse anti-actin monoclonal antibody (Chemicon, Temecula, CA, USA) was used as an internal control. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG was utilized as the secondary antibody (Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA), and signals were detected using Immobilon Western (Millipore). Membranes were exposed to standard X-ray film and densitometric quantification was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). All experiments were repeated at least five times.
7
Western Blot Protein Analysis Protocol
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Protein lysates were extracted in the SDS-lysis buffer (0.5 mM EDTA, 20 mM HEPES, 2% (w/v) SDS pH 7.9). Samples were boiled to 95°C for 5 min and DNA was sheared using a 26G needle before being stored at −20°C for further processing. Equal amounts of protein lysates (10–30 μg) were loaded into 4%–15% precast gels (Bio-Rad) and ran using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in Tris-glycine-SDS running buffer. Separated proteins were then transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore IPVH00010) using the Mini-PROTEAN III system (Bio-Rad). After transfer, membranes were blocked with 5% milk in TBS-T blocking buffer at RT for 1 h and then incubated with primary antibodies diluted in blocking buffer at 4°C overnight. The following morning, the membranes were washed three times with TBS-T then incubated with mouse or rabbit horseradish peroxide-bound secondary antibodies followed by three washes with TBS-T. The protein was visualized using the Western Lighting Plus ECL kit (PerkinElmer). Images were acquired using ChemiDoc Touch system (Bio-Rad) and processed by Image Lab software (Bio-Rad). The primary and secondary antibodies are listed in Table S2 .
8
Western Blot Protein Detection Protocol
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Equivalent protein amounts were denaturated in loading buffer at 1X final concentration at 95 °C for 5 min, separated on SDS-PAGE gels (BioRad 4–15% TGX pre-cast gels) before transfer overnight onto Immobilon-P polyvinylidene difluoride (PVDF, Millipore) or nitrocellulose (0.45 μm pore, Bio-Rad or Protran, GE Healthcare) membranes. Staining with AdvanStain Iris (Advansta), or Ponceau S, controlled homogeneous loading and prestained protein ladder allowed cutting the membrane into stripes to simultaneously blot multiple antibodies. Membranes were blocked for 60 min with 5% non-fat dry milk in PBS-T buffer, incubated as necessary with primary antibody diluted in PBS-T containing 1% or 2.5% bovine serum albumin (immunoglobulin- and lipid-free fraction V, Sigma-Aldrich) and washed 3 times with PBS-T; membranes were incubated for at most 1 h with HRP-conjugated secondary antibodies in PBS-T and washed three times with PBS-T. A list of antibodies and conditions of use is provided in Table 2 . Immuno-blots were either visualized using autoradiography films together with enhanced chemiluminescence (WesternBright ECl, Advansta), or by imaging membranes on an Amersham Imager 600 (GE Healthcare) with HyGLO Quick Spray (Denville Scientific).
9
Western Blot Analysis of NANOS1 and TUBB3
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Whole cell lysates were prepared in RIPA buffer. Western blot was performed by standard procedures using polyvinylidene fluoride membranes (Immobilon-P polyvinylidene difluoride, Millipore). The following antibodies were used: rabbit anti-NANOS1 (Abcam AB65203), 1:100; mouse monoclonal anti TUBB3 (Sigma-Aldrich T8660), 1:400; and HRP-conjugated secondary antibodies (Jackson 715-035-150 and 111-035-144), 1:10,000. ECL Prime (GE Healthcare) was used.
10
Brush Border Membrane Vesicle Immunoblotting
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Brush border membrane vesicle (BBMV)s prepared using the Ca 2+ precipitation method were obtained from mouse intestine and kidneys and used for immunoblotting as described previously (21) (22) (23) . Protein samples were heated at 95°C for 5 min in a sample buffer with the presence of 2-mercaptoethanol and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred by electrophoresis to Immobilon-P polyvinylidene difluoride (Millipore, Billerica, MA, USA) and treated with diluted antibodies. Signals were detected using Immobilon Western (Millipore).
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