Total protein samples were lysed in RIPA buffer (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40 and 50 mM Tris-HCl, pH 7.6) containing a protein inhibitor cocktail (Roche, Mannheim, Germany). After separation on 8% polyacrylamide gels and transfer to a 0.45 μm membrane (Millipore, Billerica, MA, USA), the proteins were detected using anti-MRP1 (Abcam, Hong Kong, China) and anti-GAPDH (Sigma) antibodies.
Anti mrp1
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-MRP1 is a laboratory reagent used for the detection of the Multidrug Resistance Protein 1 (MRP1) in various research applications. MRP1 is an ATP-binding cassette transporter that plays a role in the efflux of various substances from cells. This product is intended for research use only and its specific applications should not be extrapolated.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase conjugated secondary antibody, by Bio-Rad (2 mentions)
Anti mdr1, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
0.45 μm membrane, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Protein inhibitor cocktail, by Roche (1 mentions)
Vinblastine, by Merck Group (1 mentions)
Diethylstilbestrol, by Merck Group (1 mentions)
Anti abcg2, by Abcam (1 mentions)
Mk571, by Merck Group (1 mentions)
Doxorubicin chlorhydrate, by Cytiva (1 mentions)
Fumitremorgin c, by Merck Group (1 mentions)
Cyclosporine a, by Merck Group (1 mentions)
Paclitaxel, by Merck Group (1 mentions)
Sunitinib, by Pfizer (1 mentions)
Enhanced chemiluminescence, by Bio-Rad (1 mentions)
Anti β tubulin, by Santa Cruz Biotechnology (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Clarity western ecl substrate chemiluminescence detection kit, by Bio-Rad (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
10 protocols using anti mrp1
1
Protein Expression Analysis by Western Blot
2
Antibody-Based Multidrug Resistance Detection
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Mouse monoclonal anti-P-gp, anti-ABCG2 and anti-MRP1 antibodies were purchased from Abcam (Cambridge, UK). Sunitinib was obtained from Pfizer, Inc. (New York, NY, USA). Doxorubicin chlorhydrate was purchased from Amersham Pharmacia Biotech, Inc. (Uppsala, Sweden). Verapamil was obtained from Calbiochem (Billerica, MA, USA). Paclitaxel, vinblastine, cyclosporine A, fumitremorgin C, diethylstilbestrol and MK571 were purchased from Sigma-Aldrich (Saint Louis, MO, USA).
3
Protein Extraction and Western Blot Analysis
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Cells were rinsed with ice-cold lysis
buffer (50 mM, tris, 10 mM ethylenediamine tetraacetic acid (EDTA),
1% v/v Triton-X100), supplemented with the protease inhibitor cocktail
set III (80 μM aprotinin, 5 mM bestatin, 1.5 mM leupeptin, 1
mM pepstatin; Calbiochem, San Diego, CA), 2 mM phenylmethylsulfonyl
fluoride, and 1 mM Na3VO4, then sonicated and
centrifuged at 13 000g for 10 min at 4 °C.
Protein extracts (20 μg) were subjected to sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and probed with the
following antibodies: anti-P-gp (C219, Calbiochem), anti-MRP1 (Abcam,
Cambridge, U.K.), anti-CAXII (Abcam, Cambridge, U.K.), and anti-β-tubulin
(Santa Cruz Biotechnology Inc., Santa Cruz, CA), followed by a peroxidase-conjugated
secondary antibody (Bio-Rad Laboratories). The membranes were washed
with tris-buffered saline-Tween 0.1% v/v solution, and the proteins
were detected by enhanced chemiluminescence (Bio-Rad Laboratories).
buffer (50 mM, tris, 10 mM ethylenediamine tetraacetic acid (EDTA),
1% v/v Triton-X100), supplemented with the protease inhibitor cocktail
set III (80 μM aprotinin, 5 mM bestatin, 1.5 mM leupeptin, 1
mM pepstatin; Calbiochem, San Diego, CA), 2 mM phenylmethylsulfonyl
fluoride, and 1 mM Na3VO4, then sonicated and
centrifuged at 13 000g for 10 min at 4 °C.
Protein extracts (20 μg) were subjected to sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and probed with the
following antibodies: anti-P-gp (C219, Calbiochem), anti-MRP1 (Abcam,
Cambridge, U.K.), anti-CAXII (Abcam, Cambridge, U.K.), and anti-β-tubulin
(Santa Cruz Biotechnology Inc., Santa Cruz, CA), followed by a peroxidase-conjugated
secondary antibody (Bio-Rad Laboratories). The membranes were washed
with tris-buffered saline-Tween 0.1% v/v solution, and the proteins
were detected by enhanced chemiluminescence (Bio-Rad Laboratories).
4
Quantitative Protein Analysis from Kidney Tissue
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Proteins were extracted from kidney and WT frozen tissues in RIPA buffer (150 mM NaCl, 1% (v/v) NP40, 50 mM Tris-HCl pH 8.0, 0.1% (v/v) SDS, 1 mM EDTA and 0.5% (w/v) deoxycholate) supplemented with 10 mM NaF and 2 mM NaOv. Samples were incubated for 20 min on ice and centrifuged for 15 min at 13000 r.p.m. at 4°C. Supernatants were collected and quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). Equivalent amounts of proteins were resolved by SDS polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Darmstadt, Germany). Immunoblotting was performed using the following antibodies: anti-ABCB4 (Abcam-ab184878) overnight at 1:200 dilution; anti-MRP1 (Abcam-ab137406) overnight at 1:200 dilution; anti-β-actin (A5441 clone AC-15) for 1.5 h at 1:10000 dilution; anti-rabbit IgG, HRP (Cell Signaling, ref#7074) for 1 h at 1:10000; anti-mouse IgG-HRP (Cell Signaling, ref#7076) for 1 h at 1:10000. Protein bands were visualized using the Clarity Western ECL Substrate chemiluminescence detection kit (Bio-Rad, ref#170-5060). All of the antibodies were previously analyzed for antigen specificity in our laboratory, all conditions being optimized for specific antigen detection, with elimination of nonspecific reactivity.
5
Protein Expression Analysis of MDR Factors
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Total cellular protein was extracted using ice-cold RIPA lysis buffer (50 mM Tris–HCl, pH = 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate) containing 1× complete protease inhibitor cocktail (EDTA-free, Sigma-Aldrich). Protein extracts were electrophoresed on a 10% SDS polyacrylamide gel and transferred to the Immobilon-P PVDF membrane (Millipore, Billerica, MA, USA). The following primary antibodies were used: anti-multidrug resistance-related protein 1 (anti-MRP1, Abcam, Cambridge, UK; dilution 1 : 1000), anti-multidrug resistance 1 (anti-MDR1, Abcam; dilution 1 : 2000), anti-Myb (Abcam; dilution 1 : 1000) and anti-β-actin (Abcam; dilution 1 : 5000). Horseradish peroxidase-conjugated IgG (Abcam; dilution 1 : 5000) was used a second antibody. Protein bands were detected using ECL reagent (GE Healthcare Technologies, Waukesha, WI, USA) and analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
6
Western Blot Analysis of Drug Resistance Proteins
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Total proteins were extracted from cultured cells using SDS lysis buffer (Beyotime, Shanghai, China). Equal amounts of protein were analyzed by 8% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% non-fat milk at room temperature for 1 h, membranes were incubated with the following primary antibodies overnight at 4°C: anti-P-gp (1:1000; Cell Signaling Technology, United States), anti-MRP1 (1:500; Abcam, United States), anti-BCRP (1:500; Millipore, United States), anti-α-tubulin, and anti-β-actin (1:5000; Proteintech Group, United States). The appropriate peroxidase-conjugated antibodies (anti-mouse or anti-rabbit) were then incubated with the membranes at room temperature for 2 h. Reactive bands were detected using ECL-Plus reagent (Merck Millipore, Darmstadt, Germany), and the band intensity was quantified using a Bio-Rad 2000 gel imaging system equipped with QUANTITY ONE software (Bio-Rad Laboratories, Hercules, CA, United States).
7
Western Blot Analysis of Stemness and Resistance Markers
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Total protein from cells were extracted using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.). The protein concentration was determined with the application of a BCA assay kit (Beyotime Institute of Biotechnology). Afterwards, 10% SDS-PAGE gels were prepared to separate proteins (30 µg each lane), and then the latter were transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore). Then, the PVDF membrane was fixed with 5% skimmed milk powder at room temperature for 1.5 h. PVDF membranes were incubated with primary antibodies, including anti-CD133 (1:1,000; cat. no. ab222782; Abcam), anti-MDR1 (1:1,000; cat. no. ab235954; Abcam), anti-MRP1 (1:1,000; cat. no. ab3368; Abcam) and anti-GAPDH (1:1,000; cat. no. ab8245; Abcam), and were removed the second day. In addition, goat anti-mouse IgG antibody (1:5,000, cat. no. AP127F; Sigma-Aldrich; Merck KGaA) were prepared for incubation with PVDF membranes at room temperature for 1.5 h. The protein bands were visualized and analyzed using a chemiluminescence system (Bio-Rad Laboratories, Inc.) and semi-quantified using ImageJ software (version 1.46; National Institutes of Health).
8
Methylation Inhibitor Sensitization of Cancer Cells
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2-ME (purity >98.0%) was obtained from Zhengzhou University (Zhengzhou, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Roswell Park Memorial Institute (RPMI)-1640 cell culture media were bought from Gibco Invitrogen. Dimethyl sulfoxide (DMSO) was obtained from Tianjin Deen Chemical Reagent Co., Ltd. (Tianjin, China). The water was pretreated with a Milli-Q-plus system (Millipore, Bedford, MA, USA). An annexin V-fluorescein isothiocyanate (FITC) propidium iodide (PI) apoptosis detection kit was purchased from Keygen Biotech. Co. (Cat No. KGA106, Nanjing, China). 5-Aza-2’-deoxycytidine (5-Aza-dC) was purchased from Sigma Aldrich Chemical Company (St. Louis, MO, USA). The nuclear-cytosol extraction kit and bicinchoninic acid (BCA) protein assay kits were purchased from Applygen Technologies Inc. The EpiQuiKTM DNA methyltransferase activity/inhibition assay kit was purchased from Epigentek. The human polyclonal anti-DNMT1, anti-P-gp, anti-BCRP, and anti-MRP1 antibodies were purchased from Abcam; β-actin antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).
9
Protein Analysis of Esophageal Cancer
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The total proteins from esophageal cancer tissues or cultured esophageal cancer cells were extracted using a Total Protein Extraction kit (#C006225; Sangon Biotech) according to the manufacturer's instructions. Next, ∼25 μg of protein from each extract was added to a protein loading buffer and boiled at 100 °C for 5 min. The protein samples were then separated by 12% SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis; Sangon Biotech), and the protein bands were transferred onto PVDF (polyvinylidene fluoride) membranes, which were subsequently blocked with 5% lipid-free milk solution. The membranes were then incubated overnight with diluted primary antibodies at 4 °C, washed 3 times with TBST, incubated for 1 to 2 h with diluted secondary antibodies at room temperature, and finally developed with ECL Western Blotting Substrates (#32109; Thermo Fisher Scientific) as instructed by the manufacturer. The primary antibodies used for western blotting were anti-ATP7A (#ab13995; Abcam), anti-MRP1 (#14685; CST), anti-MRP1 (#14685; Abcam), anti-ABCG1 (#ab218528; Abcam), anti-ABCA1 (#ab18180; Abcam), anti-VEGF (#2463; CST), anti-Caspase 3 (#ab2302; Abcam), and anti-GAPDH (#5174; CST).
10
Plasma Membrane and Mitochondrial Protein Analysis
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Plasma-membrane-extracts were isolated by the biotination method, using the Cell Surface Protein isolation kit (Thermo Fisher Scientific Inc., Waltham, MA), as reported elsewhere 12 using an anti-pancadherin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) to check equal protein loading. Mitochondrial extracts were prepared as reported elsewhere, 8 using an anti-porin (Abcam) antibody to check equal loading of proteins. 50 µg proteins from plasma-membrane or 10 µg proteins from mitochondrial extracts were separated by SDS-PAGE and probed with the following antibodies: anti-Pgp (Santa Cruz Biotechnology), anti-MRP1 (Abcam, Cambridge, UK), and anti-BCRP (Santa Cruz Biotechnology). To analyze the presence of nitrated proteins, the mitochondrial extract was subjected to immunoprecipitation using a rabbit polyclonal anti-nitrotyrosine antibody (Millipore, Bedford, MA). Immunoprecipitated proteins were separated by SDS-PAGE and probed with anti-Pgp, anti-MRP1, and anti-BCRP antibodies. After overnight incubation, the membrane was washed with PBS-Tween 0.1% v/v and treated for 1 h with a peroxidase-conjugated secondary antibody (Bio-Rad Laboratories). The membrane was washed with PBS-Tween 0.1% v/v, and proteins were detected by enhanced chemiluminescence (Immun-Star, Bio-Rad Laboratories).
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