All samples were lysed in RIPA buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (GE Healthcare Life Science, Little Chalfont, U.K.). About 5% (w/v) non-fat dry milk in Tris-buffered saline containing Tween 20 was used to seal membranes. Then, membranes were cultured all night with specific primary antibodies, including anti-HDAC2 (ab32117; 1/2000, Abcam, Cambridge, U.S.A.), anti-GAPDH (ab22555; 1/1000, Abcam) and anti-PLIN1 (ab3526; 1/1000, Abcam). Horseradish peroxidase-conjugated secondary antibody was incubated at room temperature (25°C) for 1 h. Finally, membranes were visualized via exposure to Immobilon Western Reagents (Millipore, Billerica, MA, U.S.A.).
Immobilon western reagent
Manufactured by Merck Group
Sourced in United States, Japan
Immobilon Western reagent is a detection reagent used in Western blotting analysis. It is designed to visualize and quantify specific proteins that have been separated by gel electrophoresis and transferred to a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Molecular imager chemidoc xrs, by Bio-Rad (4 mentions)
Chemocam imager, by Intas (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (3 mentions)
Mini trans blot apparatus, by Bio-Rad (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Complete mini, by Roche (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab32117, by Abcam (1 mentions)
Ab22555, by Abcam (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Ab3526, by Abcam (1 mentions)
Chemcam imager, by Intas (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Mouse monoclonal anti flag m2 antibody, by Merck Group (1 mentions)
Anti mouse igg antibody, by Cell Signaling Technology (1 mentions)
Mouse anti his6 antibody, by Roche (1 mentions)
Anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Mouse anti ha antibody, by Santa Cruz Biotechnology (1 mentions)
Mouse anti gapdh antibody, by Merck Group (1 mentions)
21 protocols using immobilon western reagent
1
Western Blot Analysis of HDAC2, GAPDH, and PLIN1
2
Western Blot Analysis Protocol
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Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% fat-free dried skimmed milk in TBS-T (10 mM Tris HCl pH 7.6, 150 mM NaCl, 1% Tween 20) and probed with the indicated primary antibodies in 5% BSA in TBS-T at 4°C overnight. After washing, membranes were incubated with secondary peroxidase-conjugated antibodies (1:2,500) diluted in 5% fat-free dried skimmed milk. The Immobilon™ Western Reagents (Millipore Corporation, Billerica, MA, United States) and the ChemCam Imager (INTAS Science Imaging Instruments GmbH, Göttingen, Germany) were used for signal detection.
3
Western Blot Protein Detection
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Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes for 60 min (20 V, 1.0 Å). Membranes were blocked and probed with the indicated primary antibodies. After washing, the membranes were incubated with secondary peroxidase-conjugated antibodies or fluorescence-labeled secondary antibodies (1:2,500 dilution). Immobilon Western reagents (Millipore Corporation, Billerica, MA, USA) and the ChemoCam imager (Intas Science Imaging Instruments GmbH, Göttingen, Germany) or the Odyssey Fc imaging system (Li-Cor Biosciences, Bad Homburg, Germany) were used for signal detection. Control STAT3 blots were produced on separate membranes.
4
SDS-PAGE Western Blot Protocol
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Cells (1 × 106) were washed twice with PBS (phosphate buffered saline), lysed in 500 μL of 2× SDS-PAGE sample buffer (8% SDS, 40% glycerol, 0.25 M Tris-HCl (pH 6.8), 2% BPB and 5% β-mercaptoethanol), and boiled for 10 min. Following SDS-PAGE, the gel was subjected to western blotting and visualized using a LAS3000 mini (Fujifilm Co., Tokyo, Japan) and Immobilon Western Reagents (Millipore). Antibodies used in the experiments were as follows: mouse monoclonal (M2) anti-FLAG antibody (dilution; 1:5000) was purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA); the rabbit polyclonal anti-Myc antibody (1:5000), the mouse anti-GST antibody (1:5000), and the mouse anti-HA antibody (1:5000) were obtained from Santa Cruz biotechnology; the mouse anti-(His)6 antibody (1:500) was obtained from Roche; the mouse anti-GAPDH antibody (1:5000) was from Millipore. Secondary antibodies conjugated with horseradish peroxidase (anti-Rabbit IgG antibody (1:5000) and anti-Mouse IgG antibody (1:5000, except for the case with anti-GAPDH (1:10,000))) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
5
Western Blot Analysis Procedure
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All samples were lysed in RIPA buffer and separated by SDS-PAGE and transferred to NC membranes (GE Healthcare Life science, Lot. G9597136). The 5% (w/v) no-fat dry milk in TBST was used to block non-specific binding sites. The blocked membranes were incubated with the specific primary antibodies diluted in 5%BSA (Sigma-Aldrich) with 0.05% sodium azide for overnight on shacking bed at 4°C. HRP-conjugated secondary antibody was incubated at room temperature for 1 h. Finally, the membranes were visualized by exposure with Immobilon Western Reagents (Millipore, Lot.15015B4).
6
Quantification of Kidney Transporter Proteins
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Kidney proteins from the unclipped kidney were isolated in RIPA buffer with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, US). Total protein quantification was performed using the Lowry method [37]. Equal protein amounts were loaded and separated by electrophoresis on glycine SDS-PAGE gels. Proteins were transferred to 0.45 µm PVDF membranes (Millipore, Burlington, MA, USA) and blocked for 1 h with 5% non-fat dry milk in 1X PBS-T buffer. After blockage, the membranes were incubated overnight with the following primary antibodies: (1) anti-NHE3 (a generous gift from Dr. Peter Aronson (Yale University, New Haven, CT, USA), (2) anti-NCC (a generous gift from Dr. Alicia McDonough), (3) anti-ENaC γ (Alamones Labs, Jerusalem, IL), (4) anti-ENaC β (Alamones Labs, Jerusalem, IL), (5) anti-GAPDH (Abcam, Cambridge, MA, USA), or (6) anti-actin (Merck, Darmstadt, DE). They were also incubated for 1 h with secondary HRP-conjugated antibodies (Abcam). The signal was measured using chemiluminescence with Immobilon Western reagents (Millipore, Burlington, MA, USA). Band densitometry was performed using ImageJ software version 1.53 (NIH, Bethesda, MD, USA).
7
Western Blot Analysis of PI3K/Akt Signaling
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Whole-cell lysates from RPMI-8226 cells were prepared. The amount of cellular protein was measured by the method proposed by Lowry et al31 (link) and stored at −80°C prior to use. Twenty microgram of protein of each sample was separated on SDS-PAGE gels as previously described.32 (link) After electrophoresis, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with Tris-buffered saline 0.05% Tween-20 containing 5% skim milk or 3% bovine serum albumin (Sigma, St Louis, MO, USA), blots were incubated at 4°C overnight with the primary rabbit polyclonal or monoclonal antibodies against human PI3K (1:1,000), Akt (1:500), phospho-Akt (pSer473, 1:500), Caspase-3 (1:2,000), Bcl-2 (1:500), and Bax (1:500) (Cell Signaling Technology, Danvers, MA, USA), respectively. Membranes were reprobed with anti β-actin antibody (1:1,000; Sigma, St Louis, MO, USA) as loading control. Horseradish peroxidase (HRP)-conjugated sheep anti-rabbit or mouse immunoglobulins were used as a secondary antibody (Sigma, St Louis, MO, USA). The bounded antibody was detected by enhanced chemiluminescence using Immobilon Western reagents (Millipore, Billerica, MA, USA) and exposed to X-ray film according to the manufacturer’s protocol.
8
CXCR7 Signaling Pathway Analysis
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RPMI media was from Invitrogen (Carlsbad, CA, USA). Enhanced chemiluminescence (ECL) reagents and nitrocellulose membrane Hybond-C extra were from Amersham Pharmacia Biotech (Cologno Monzese, Milano, Italy), Immobilon Western reagents and ECM cell adhesion array kit were from Millipore Corporation (Billerica, MA, USA).
Recombinant CXCL12 was purchased from R&D (Minneapolis, MN, USA). CCX733, CCX771 and CCX704 were kindly provided by Dr. Mark Penfold (Chemocentryx, Mountain View, CA, USA). Anti-CXCR7 antibody used for immunoprecipitation was from Biolegend (San Diego, CA, USA). Antibodies against CXCR4, β-arrestin 2, PARP and anti-CXCR7 antibodies used for Western blotting were from Abcam (Cambridge, United Kingdom). Immunohistochemistry was performed with an anti-CXCR7 Ab purchased from Proteintech (Rosemont, IL, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), arsenic thrioxide (AT), monoclonal antibodies against β-actin and vinculin were purchased from Sigma (St. Louis, MO, USA). All other reagents were of analytical grade.
Recombinant CXCL12 was purchased from R&D (Minneapolis, MN, USA). CCX733, CCX771 and CCX704 were kindly provided by Dr. Mark Penfold (Chemocentryx, Mountain View, CA, USA). Anti-CXCR7 antibody used for immunoprecipitation was from Biolegend (San Diego, CA, USA). Antibodies against CXCR4, β-arrestin 2, PARP and anti-CXCR7 antibodies used for Western blotting were from Abcam (Cambridge, United Kingdom). Immunohistochemistry was performed with an anti-CXCR7 Ab purchased from Proteintech (Rosemont, IL, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), arsenic thrioxide (AT), monoclonal antibodies against β-actin and vinculin were purchased from Sigma (St. Louis, MO, USA). All other reagents were of analytical grade.
9
Western Blot Protocol for Protein Detection
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Proteins were separated by SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) or nitrocellulose membranes. Membranes were blocked and probed with the indicated primary antibodies. After washing, membranes were incubated with secondary peroxidase-conjugated antibodies (1:2.500 dilution) or fluorescence-labeled secondary antibodies (1:10.000 dilution). The ImmobilonTM Western Reagents (Millipore Corporation, Billerica, MA, USA) and the ChemoCam Imager (INTAS Science Imaging Instruments GmbH, Göttingen, Germany) or the Odyssey Fc Imaging System (LI-CORE Biosciences, Bad Homburg, Germany) were used for signal detection. Control STAT3 and ERK blots were produced on a separate membrane using the same samples.
10
Western Blot Analysis of Protein Phosphorylation
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Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) or nitrocellulose membranes. Membranes were blocked and probed with the indicated primary antibodies. After washing, membranes were incubated with secondary peroxidaseconjugated antibodies or fluorescence-labeled secondary antibodies (1:10.000 dilution). The Immobilon TM Western Reagents (Millipore Corporation, Billerica, MA, USA) and the ChemoCam Imager (INTAS Science Imaging Instruments GmbH, Göttingen, Germany) or the Odyssey Fc Imaging System (LI-CORE Biosciences, Bad Homburg, Germany) were used for signal detection. Control STAT3 blots were produced on the same membrane either following stripping or simultaneously to pSTAT3 imaging using the Odyssey Fc Imaging System.
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