Six commercially available anti-ABCG2 antibodies were purchased from five vendors. The mouse monoclonal antibody (mAb) BXP-21 was purchased from Abcam (Cambridge, UK), and the immunogen was a fusion protein composed of E. coli maltose binding protein and ABCG2 peptide (aa271-396). The mouse mAb 3G8 was purchased from Abnova (Taipei City, Taiwan), and the immunogen was a recombinant protein corresponding to human ABCG2 (aa153-360). The mouse mAb 6D171 was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA), and the immunogen was ABCG2 of human origin (aa271-396). The rabbit polyclonal antibody (pAb) B7185 was purchased from Sigma-Aldrich (St. Louis, Missouri, USA), and the immunogen was a synthetic peptide corresponding to aa150-167 of ABCG2 with an added C-terminal cysteine conjugated to keyhole limpet hemocyanin (KLH). The rabbit pAb TA324234 was purchased from Origene (Rockville, USA), and the immunogen was a synthetic peptide directed toward the N-terminal of human ABCG2 within the region aa50-99. The rabbit pAb TA332085 was also purchased from Origene, and the immunogen was a synthetic peptide corresponding to a region derived from aa609-621 of human ABCG2. The pAb TA324234 was the only antibody with an extracellular immunogen, whereas the immunogens for all of the other antibodies were from intracellular domains of ABCG2.
Bxp 21
Manufactured by Abcam
Sourced in United States, United Kingdom
BXP-21 is a laboratory equipment product manufactured by Abcam. It serves as a benchtop centrifuge for the separation and purification of biological samples. The device can accommodate various sample sizes and speeds to facilitate experimental workflows.
Automatically generated - may contain errors
Lab products found in correlation
Anti α tubulin, by Merck Group (4 mentions)
Ecl kit, by Merck Group (3 mentions)
Hdac6, by Merck Group (2 mentions)
Anti hdac6, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Abcam (1 mentions)
α tubulin, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti mouse immunoglobulin g igg, by Abcam (1 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Anti α tubulin clone b512, by Merck Group (1 mentions)
Irdye 680rd goat anti mouse dyes, by LI COR (1 mentions)
Nunc lab tek 2 chambered, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Anti acetylated α tubulin, by Merck Group (1 mentions)
Envision system hrp kit, by Agilent Technologies (1 mentions)
Hrp conjugated goat anti mouse igg h l secondary antibody, by Abcam (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Pngase f, by Roche (1 mentions)
Image lab software version 5, by Bio-Rad (1 mentions)
Stain free technology, by Bio-Rad (1 mentions)
17 protocols using bxp 21
1
Commercially Available Anti-ABCG2 Antibody Evaluation
2
Quantification of ABC Transporter Levels
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Human cancer cells overexpressing ABCB1 or ABCG2 were treated with DMSO (control) or sitravatinib at 100 nM, 200 nM, 300 nM or 500 nM for 72 h before being harvested and subjected to SDS-polyacrylamide electrophoresis and Western blotting as described previously [49 (link)]. Primary antibodies C219 (1:3000 dilution) and BXP-21 (1:1000 dilution) were purchased from Abcam (Cambridge, MA, USA) and used to detect ABCB1 and ABCG2, respectively. Primary antibody α-tubulin (1:100,000 dilution) was used for the detection of tubulin, a positive loading control. The horseradish peroxidase-conjugated goat anti-mouse IgG (1:100,000 dilution) was purchased from Abcam (Cambridge, MA, USA) and used as secondary antibody. The enhanced chemiluminescence (ECL) kit was purchased from Merck Millipore (Billerica, MA, USA) and the signals were detected as described previously [49 (link)].
3
Immunoblotting for ABCB1, ABCG2, and HDAC6
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An immunoblot assay using antibodies C219 (#517310, Merck Millipore, Burlington, Massachusetts, USA) at 1:3000, BXP-21 (#ab3380, Abcam, Cambridge, MA, USA) at 1:15,000, anti-HDAC6 (#7558, Cell Signaling Technology, Danvers, MA, USA) at 1:1000, and anti-α-tubulin (#T6199, Sigma-Aldrich, St. Louis, MO, USA) at 1:100,000 was performed to identify ABCB1, ABCG2, HDAC6, and tubulin as a positive control for Western blotting, as described previously [56 (link)]. The horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) and anti-rabbit IgG were used as secondary antibodies, and signals were detected as described previously [53 (link)].
4
ABCG2 Expression Quantification via Immunoblot
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An immunoblot assay for ABCG2 was performed on ABCG2-overexpressing S1-M1-80 and H460-MX20 cancer cells using the BXP-21 (1:15000 dilution) antibody (Abcam, Cambridge, MA, USA), as previously described [8 (link)]. The α-tubulin (1:100,000 dilution) antibody (Sigma-Aldrich, St. Louis, MO, USA) was used to detect the positive loading control tubulin. The horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) (1:100,000 dilution) (Sigma-Aldrich, St. Louis, MO, USA) was used as the secondary antibody. Signals were detected using the enhanced chemiluminescence (ECL) kit (Merck Millipore, Billerica, MA, USA).
5
Quantifying ABCB1 and ABCG2 Protein Levels
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Immunoblotting was carried out following the standard procedure as previously described [20 (link)]. In brief, cancer cells were treated with DMSO (control) or furmonertinib at concentrations of 20 nM, 100 nM, 200 nM, or 1000 nM for 72 h. After the treatment, the cells were harvested and subjected to SDS-polyacrylamide electrophoresis (SDS-PAGE). For Western blotting, the primary antibody C219 (diluted 1:3000, Merck Millipore, Burlington, MA, USA) was used to detect human ABCB1, the primary antibody BXP-21 (diluted 1:15,000, Abcam, Cambridge, MA, USA) was used to detect human ABCG2, and the primary antibody anti-alpha tubulin (diluted 1:100,000, Sigma-Aldrich, St. Louis, MO, USA) was used as a positive loading control for tubulin. A horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) (diluted 1:100,000, Abcam, Cambridge, MA, USA) was used as the secondary antibody. Signal detection was performed using the enhanced chemiluminescence (ECL) kit (Merck Millipore, Billerica, MA, USA), as described in previous work [20 (link)].
6
Quantitative Western Blot Analysis of Drug Transporters
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The following antibodies were used. Anti-SERT rabbit polyclonal antibody/AB10514P (EMD Millipore, Billerica, MA); anti-P-gp mouse monoclonal antibody Clone 2F7 (OriGene, Rockville, MD); anti-MRP3 rabbit anti-human antibody LS-C177398 (Lifespan Biosciences Inc, Seattle, WA); anti-NET rabbit polyclonal antibody LS-C101935 (Lifespan Biosciences Inc, Seattle, WA) and anti-BCRP mouse monoclonal antibody BXP-21 (Abcam, Cambridge, MA). Loading control antibodies included: Anti-GAPDH (6C5, sc-32233, Santa Cruz Biotechnologies, Santa Cruz, CA); mouse monoclonal Grb2 (BD Biosciences, San Jose, CA), and anti-α-Tubulin clone B512 (Sigma-Aldrich Co, St. Louis, MO). Primary antibodies were diluted according to the manufacture’s suggestion (1:1,000). IRDye® 800CW Goat Anti-Rabbit and IRDye® 680RD Goat Anti-Mouse Li-COR dyes (LI-COR, Inc., Lincoln, NE) were diluted at 1:20,000. RFU readings were normalized to control housekeeping proteins (Grb2, Tubulin, or GAPDH) in qWestern-blot assays.
7
Immunofluorescence Analysis of Protein Localization
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HEK cells were seeded onto 8-well Nunc Lab-Tek II chambered (Thermo Fisher, cat. 155409) at 5 × 104 cells/well density and grown for 48 h after transfection. Stably expressing HeLa cells were seeded onto ibiTreat µ-Slide 8 Well (Ibidi, cat. 80826) at 2 × 104 cells/well density and grown for 24 h. The samples were gently washed with Dulbecco’s modified phosphate-buffered saline (DPBS) and then fixed with 4% paraformaldehyde in DPBS for 10 min at room temperature. After several washing steps, the cells were blocked for 1 h at room temperature in DPBS containing 2% bovine serum albumin, 1% fish gelatin, 0.1% Triton-X 100, and 5% goat serum (blocking buffer). The samples were then incubated for overnight at 4 °C with the primary antibody (Bxp-21, 1:200, Abcam, cat. ab3380) diluted in blocking buffer. After washing with DPBS, the cells were incubated for 1 h at room temperature with the secondary antibody (Alexa Fluor 647-conjugated goat anti-mouse, Thermo Fisher, cat. A-21235) diluted 1:250 in blocking buffer. Nuclei were stained with DAPI (1 µM final concentration). Cells were examined with Zeiss LSM 5710 laser scanning fluorescence confocal microscope (40 ×, oil immersion objective), and images were processed with ZEN 2012 software.
8
Antibody-based detection of cellular proteins
9
Immunohistochemical Profiling of Stem Cell Markers
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Antibodies against ABCG2 (BXP-21) (1 : 50; mouse monoclonal; Abcam, Cambridge, UK), CD133 (1 : 300; rabbit polyclonal; Abcam), podoplanin (1 : 50; mouse monoclonal; DAKO, Carpinteria, CA) and Ki-67 (1 : 50; mouse monoclonal; DAKO) were used in this study.
For immunohistochemical staining, serial sections of 4 μm thickness were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Endogenous peroxidase activity was blocked by immersing the sections in 3% H2O2 with methanol for 30 min. For antigen retrieval, sections were boiled in 10 mmol/L citrate buffer (pH 6.0) for 15 min in a pressure cooker. After treatment with protein block serum at room temperature, sections were covered with primary antibodies and incubated at 4°C overnight. For secondary antibody, DAKO Cytomation Envision+System-HRP kit (AEC) was used according to the manufacturer's instructions. Antibody reactions were stained with 3,3′-diaminobenzidine and counterstained with hematoxylin. For the negative control, sections were incubated with PBS instead of the primary antibodies.
For immunohistochemical staining, serial sections of 4 μm thickness were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Endogenous peroxidase activity was blocked by immersing the sections in 3% H2O2 with methanol for 30 min. For antigen retrieval, sections were boiled in 10 mmol/L citrate buffer (pH 6.0) for 15 min in a pressure cooker. After treatment with protein block serum at room temperature, sections were covered with primary antibodies and incubated at 4°C overnight. For secondary antibody, DAKO Cytomation Envision+System-HRP kit (AEC) was used according to the manufacturer's instructions. Antibody reactions were stained with 3,3′-diaminobenzidine and counterstained with hematoxylin. For the negative control, sections were incubated with PBS instead of the primary antibodies.
10
Biotin-Induced ABCG2 Protein Expression
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Protein from the untreated cells (0 h) as well as from those subjected to 100 μM biotin for the indicated times was extracted by the addition of TE buffer (0.1 M TRIS-PO4, 4% SDS, 4 mM Na-EDTA, 40% glycerol, 0.04% bromophenol blue, and 0.04% β-mercaptoethanol; materials from Sigma-Aldrich), and then the samples were sonicated. In certain experiments, when indicated, the cells were pre-treated with 1 mM 4-PBA, 2 μm MG132, or 10 nM BAF overnight prior to biotin addition. Equal amounts of the protein samples were loaded onto 7.5% polyacrylamide gels. For glycosidase treatments, cell lysates containing 20 μg of protein were subjected to 20 U Endoglycosidase H (Endo H) (Sigma, cat.11088726001) or 20 U PNGase F (Roche, cat.11365185001) according to the manufacturer’s protocols. Blots were probed with anti-ABCG2 (Bxp-21, Abcam, cat. ab3380) or anti-β-actin (Sigma, cat. A1978) primary antibodies, and subsequently developed with HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (Abcam, cat. ab97023). Detection was performed by luminography using Clarity Western ECL Substrate (Bio-Rad, cat. 1705060). For quantification, densitometry was carried out by the ImageJ software.
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