MA1-26528

antibody from Invitrogen Antibodies

Targeting: ABCB1 ABC20, CD243, CLCS, GP170, MDR1, P-gp, PGY1

Provider product page for MA1-26528

Western blot

ELISA

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [26]
Comments [0]
Validations
Western blot [3]
Other assay [10]

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Product number: MA1-26528 - Provider product page
Provider: Invitrogen Antibodies
Product name: P-Glycoprotein Monoclonal Antibody (C219)
Antibody type: Monoclonal
Antigen: Other
Description: Recommended positive controls: Cell lines or frozen tissue, such as liver or colon. MA1-26528 is expected to cross react with a wide range of mammals due to sequence homology. Store product as a concentrated solution. Centrifuge briefly prior to opening the vial. This clone recognizes an internal, highly conserved amino acid sequence (VQEALD and VQAALD) found in both protein isoforms, MDR1 P-glycoprotein (~170kDa) and MDR3 P-glycoprotein (~140kDa). In Western blot, boiling samples may reduce visibility of the protein. For flow cytometry, cells should be fixed in 3.7% formaldehyde for 10 min at room temperature to permeabilize the cell membrane. Incubate cells with 5-10 µg antibody for 30-60 min at 4ºC.
Reactivity: Human, Mouse, Rat, Bovine, Hamster, Zebrafish
Host: Mouse
Isotype: IgG
Antibody clone number: C219
Vial size: 250 µL
Concentration: 0.72 mg/mL
Storage: Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

ABCB1 protein structure - MA1-26528 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of ABCB1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR634315_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of ABCB1 was performed by loading 30 µg of Caco-2 wild type (Lane 1) and Caco-2 ABCB1 KO (Lane 2) membrane enriched extracts. The samples were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA0378BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with P-Glycoprotein Monoclonal Antibody (C219) (Product # MA126528, 1:555 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:10,000 dilution) using the iBright™ FL1500 (Product # A44115). Chemiluminescent detection was performed usingSuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to ABCB1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of P-Glycoprotein was performed by separating 30 µg of whole cell extract by 5% SDS-PAGE. Proteins were transferred to a membrane and probed with a P-Glycoprotein Monoclonal Antibody (C219) (Product # MA1-26528) at a dilution of 1:500. The HRP-conjugated anti-mouse IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-P-Glycoprotein Monoclonal Antibody (C219) (Product # MA1-26528) and due to glycosylation, 160-230 kDa band corresponding to ABCB1 was observed across cell lines tested except MCF-7 which is reported to be negative. Whole cell extracts (30 µg lysate) of Hep G2 (Lane 1), Caco-2 (Lane 2), MDCK (Lane 3) and MCF-7 (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # LC2001) by XCell SureLock™ Mini-Cell and XCell II™ Blot Module (Product # EI0002). The blot was probed with the primary antibody (4 µg/ml) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Ketogenic diet enhances neurovascular functions. ( a ) Representative cerebral blood flow (CBF) maps superimposed on structural images; color code indicates level of CBF in a linear scale. KD mice exhibited significantly higher CBF in the ( b ) ventromedial hypothalamus. Data are presented as mean +- SEM, *** p < 0.001. ( c ) Western blot (WB) images for mTOR, P-gp, and eNOS from the cortical vasculature, beta-Actin was used as loading control. ( d ) The corresponding values of the levels of protein expression. WB data from KD mice were normalized to beta-Actin and compared to the control mice (100%), * p < 0.05, ** p < 0.01, *** p < 0.001. ( e ) Representative confocal images showing increased luminal accumulation of NBD-CSA fluorescence in brain capillaries isolated from KD mice compared to control mice, indicating higher P-gp transport activity. Corresponding quantitative fluorescence data; images are shown in arbitrary fluorescence units (scale 0-255). Data are mean +- SEM for 10 capillaries from one preparation of 10 mice per group, *** p < 0.001. mTOR: mechanistic target of Rapamycin; P-gp: P-glycoprotein; eNOS; endothelial nitric oxide synthase.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Specialized EPDC's BBB characteristics. ( A ) Quantitative RT-PCR expression analysis of specific BBB genes at day 14 of culture, in EPDCs alone (purple), with astrocytes (blue) and hCMEC-D3 (green) (qRT-PCR were performed on EPDCs RNA isolated from 6 BBB differentiation independent experiences, each individual experience was performed in triplicate). ( B ) Western blot of P-gp, GLUT-1 and OCCL in EPDCs alone (purple) or with astrocytes (blue) at day 14. Densiometric quantification was performed for each immunoblot using ImageJ software. ( C - L ) VE-CAD, ZO1, CL3, CL5 and OCCL immunofluorescence staining in EPDCs alone ( C - G ) or with astrocytes ( H - L ). Arrows show continuous junctions. ( M ) Permeability for LY (0.457 kDa) and Dextran-FITC (4 and 70 kDa) in EPDCs alone (purple) or with astrocytes (blue) and in hCMEC/D3 (green) (EPDC permeability to dextran-FITC molecules was performed once in triplicate). ( N ) Accumulation of Calcein into EPDCs alone or with astrocytes, in the presence (hatched area) or absence of Verapamil (**p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Caloric restriction enriches vascular signaling markers and shifts metabolism in young mice ( A ) Western blotting (WB) of mTOR, eNOS, P-gp and GLUT1 from the cortical vasculature, beta-Actin was used as loading control; corresponding values of ( B ) mTOR, ( C ) eNOS, ( D ) P-gp, and ( E ) GLUT1 between the young AL and CR mice. All the WB data were normalized to beta-Actin and compared to young AL (100%). ( F ) Blood glucose and ( G ) Blood Ketone levels of the mice. * p < 0.05; ** p < 0.01; *** p < 0.001; AL: ad libitum; CR: caloric restriction.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Protein expression profile in KB/ATO cells and parental KB-3-1 cells. Expression level of (A) P-gp, (B) MRP1, (C) BCRP and (D) ABCB6 were shown in western blot results. (E) RT-PCR was performed to confirm the mRNA levels of ABCB6. Cell lysate from HEK/ABCB1, HEK/ABCC1 and HEK/ABCG2 was used as positive control for P-gp, MRP1 and BCRP respectively. Fig. 4

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 10 Differential expression of ABC transporters in MES-SA and MES-SA/Dx5 to Pituranthos chloranthus (PC) and Teucrium ramosissimum Desf. (TR) extracts. ( A ) Immunoblot analysis of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance proteins (MRP1, MRP2, and MRP3) after a 72 h treatment with vehicle (dimethyl sulfoxide, DMSO; Control, Co) or indicated concentrations of PC and TR in MES-SA cells. Heat shock cognate 70 (HSC-70) was used as the loading control. A representative blot from three independent experiments is shown. ( B ) Densitometric quantification of Western blotting obtained in (A). Data are expressed as mean fold induction +- Standard Error of the Mean (SEM) of three independent experiments; p -values were determined by a one-way ANOVA followed by Tukey's multiple comparison test. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to control (Co) condition. F values of ANOVA tests (significant results): MRP2 expression, F = 15.53 and F = 28.46 for PC and TR, respectively; BCRP expression, F = 10.18 for TR. ( C ) Immunoblot analysis of P-gp, BCRP, MRP1, MRP2, and MRP3 after a 72 h treatment with vehicle (dimethyl sulfoxide, DMSO; Control, Co) or indicated concentrations of PC and TR in MES-SA/Dx5 cells. HSC-70 was used as the loading control. A representative blot from three independent experiments is shown. ( D ) Densitometric quantification of Western blotting obtained in ( C ). Data are expressed as mean fold inductio

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. P-gp and MRP2 expression in different cell lines. (A) Protein expression of P-gp was detected by western blot. (B) Quantification of P-gp by scanning of the protein bands in A. (C) Protein expression of MRP2 was detected by western blot. (D) Quantification of MRP2 by scanning of the protein bands in C. (E) mRNA expression of P-gp in MDCK, MDCK-MDR1, LLC and LLC-MDR1 cells. (F) mRNA expression of MRP2 in MDCK-WT and MDCK-MRP2 cells. Relative mRNA and protein levels of P-gp or MRP2 were scaled to the mean relative expression level of b-actin. The relative levels of P-gp or MRP2 in wild-type cells were regarded as 1. (G) Immunostaining of P-gp in wild type and MDR1 -transfected cells. Experiments were performed in triplicate, and values are shown as mean +- SEM ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Biochemical characterization of rat liver subcellular fractions and Pgp localization in endolysosomal-enriched fractions. Rat liver subcellular fractionation was performed by low speed centrifugation and subsequent density gradient ultracentrifugation. Recovery and purity of the cell organelles from collected gradient fractions 1-10 (top to bottom) were analyzed by separation of equal protein amounts (5 ug) of each fraction and whole-cell homogenate (H) and post-nuclear supernatant (PNS) by SDS-PAGE and immunoblotting. Homogenate and PNS data were similar; thus, PNS is shown here for only some proteins. Endolysosomes were detected using the LAMP-2 and Rab7 marker proteins and the luminal lysosomal protease cathepsin D (CatD). EEA1 was used as a marker for early endosomes. The purity of the endolysosomal fractions was assessed by organelle markers for mitochondria (VDAC), canalicular plasma membrane (ABCB4), and ER (calnexin). ( A ) Representative Western blots showing the distribution of the different organelle protein markers and Pgp in the gradient fractions. Some fractions (mainly 2 and 3) contained low protein amounts and thus could not be characterized for the presence of all marker proteins for each individual gradient. Therefore, marker quantification in the gradient fractions was repeatedly performed for different fractionations. ( B ) Quantification of the protein band intensities for the marker proteins in the different gradient fractions. Immunoblotting revealed re

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Detection of Pgp in the membrane fraction of F1. The endolysosome-enriched fraction was separated into soluble (luminal) and membrane subcompartments and analyzed by Western blotting for Pgp localization ( A ). The organelle membranes were ruptured applying two different methods in comparison, either repeated freeze-thaw (FT) cycles or a hypoosmotic shock (HS). The pellet (P) enriched in membrane proteins and the supernatant (S) containing soluble luminal proteins were analyzed by Western blotting (5 ug total protein/lane of P and corresponding amount of S). Liver homogenate (H), post-nuclear supernatant (PNS), and fraction 1 (F1) (5 ug total protein each) were analyzed for comparison. ( B ) LAMP-2 was used as a marker protein for the membrane fraction, whereas cathepsin D (CatD) served as a control protein for the soluble endolysosomal protein fraction.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 P-glycoprotein (P-gp) protein expression levels are decreased and P-gp ubiquitination levels are increased in brain capillaries from Alzheimer's disease (AD) patients. (A) Representative image of a P-gp-immunostained (green) brain capillary isolated from brain tissue of a cognitive normal individual (CNI); nuclei were counterstained with DAPI (blue). (B) Negative control (no primary antibody): (B1 ; green channel); (B2 ; blue channel); (B3 ; overlay of green and blue channel) and (B4 ; transmitted light channel). (C) Representative WES(tm) image and (D) electropherogram showing reduced P-gp (blue shaded area) protein expression levels in brain capillaries isolated from human brain tissue (frontal cortex) of AD patients ( n = 3) vs. CNIs ( n = 3). (E) Overlay of electropherograms displayed in (D) show a reduction in the area under the curve (AUC) that represents P-gp protein expression levels (blue shaded area) in brain capillaries from AD patients (red line) relative to CNI (blue line). In contrast, beta-actin levels (orange shaded area) in brain capillaries from AD patients (red line) and CNI (blue line) were the same. (F) Western blot showing that ubiquitin levels in P-gp-immunoprecipitates are increased in capillaries from AD patients compared to those from CNI.