Ab24102

Manufactured by Abcam

Sourced in United States, United Kingdom

Ab24102 is a laboratory equipment product offered by Abcam. It serves as a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

8 protocols using ab24102

Immunofluorescence and Western Blot Analysis

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For immunofluorescence assay, cells were grown on glass slices and fixed in 4 % formaldehyde for 10 min, permeabilized through 0.3 % Triton X-100. Then the slices were blocked in goat serum for 15 min, 37°C and incubated overnight at 4°C with anti-E-cadherin (1:80, Bioworld, MN, USA), anti-N-cadherin (1:80, Bioworld, MN, USA). Samples were washed three times before incubated with goat TRITC labeled secondary antibody (1:70, Bioworld, MN, USA) at 37°C for 1 h. DAPI (Genview Inc, Shanghai, China) was used for counterstaining. Fluorescence was visualized with a microscope under ×400 magnification.
Total protein was extracted using RIPA Lysis Buffer (Beyotime, China) and PMSF (Sigma-Aldrich). The proteins were transferred to NC membranes (Millipore Corp, MA USA) using the TransBlot System (Bio-Rad, CA, USA). The membranes were blocked in 5% w/v non-fat milk in TBS and incubations were performed overnight at 4°C. The membranes were then washed using TBST and incubated with secondary antibodies (1:10000, IRDye Goat IgG, LI-COR Bioscience, NE USA) for 1h at room temperature. Protein staining was detected using the Odyssey Imaging System (LI-COR Biosciences, NE USA). The following primary antibodies were used: GAPDH (1:10000, Proteintech Group, Chicago USA), ABCC1 (1:100, Abcam, ab24102), ABCC2 (1:100, Abcam, ab3373), P-gp1 (1:2000, Abcam, ab129450).

Sun L., Ke J., He Z., Chen Z., Huang Q., Ai W., Wang G., Wei Y., Zou X., Zhang S., Lan P, & Hong C. (2017). HES1 Promotes Colorectal Cancer Cell Resistance To 5-Fu by Inducing Of EMT and ABC Transporter Proteins. Journal of Cancer, 8(14), 2802-2808.

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Quantifying Apoptosis Pathways in Cancer Cells

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Dox, cisplatin, 5-fluorouracil, and paclitaxel were purchased from Sigma-Aldrich (St. Quentin Fallavier, France) and prepared in dimethyl sulfoxide (DMSO). Anti-Cyclin A antibody (sc-751; 1:1000), Cyclin B (sc-752; 1:1000), Cyclin E (sc-481; 1:1000), Cdk1 (sc-54; 1:1000), Cdk2 (sc-6248; 1:500), and HSC-70 (sc-7298; 1:1000) were obtained from Santa Cruz Biotechnology (Nanterre, France). Anti-PARP antibody (#9542; 1:1000), Caspase 3 (#9662; 1:1000), Caspase 8 (#4790; 1:1000), Caspase 9 (#9502; 1:1000), BCRP (#42078; 1:1000), and MRP2 (#4446; 1:1000) were purchased from Cell Signaling Technology (Ozyme, Saint-Cyr-l’École, France). Anti-MRP1 antibody (ab24102; 1:50) and MRP3 (ab3375; 1:50) were obtained from Abcam (Paris, France). Anti-P-gp antibody (#MA1-26528; 1:1000) was obtained from Invitrogen/Thermo Fisher Scientific (Paris, France).

Lahmar A., Mathey A., Aires V., Elgueder D., Vejux A., Khlifi R., Sioud F., Chekir-Ghedira L, & Delmas D. (2021). Essential Oils, Pituranthos chloranthus and Teucrium ramosissimum, Chemosensitize Resistant Human Uterine Sarcoma MES-SA/Dx5 Cells to Doxorubicin by Inducing Apoptosis and Targeting P-Glycoprotein. Nutrients, 13(5), 1719.

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Immunohistochemical Analysis of NRF2, MRP1, and PIDD

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For immunohistochemical staining, deparaffinized sections were processed for antigen retrieval by heating in sodium citrate buffer (pH 6.0). The following panel of primary antibodies was used: (1) NRF2 (1:100, ab76026, Abcam, Inc.), (2) MRP1 (1:50, ab24102, Abcam, Inc.), and (3) PIDD (1:100, sc-32161, Santa Cruz Briotechnology, Inc.). The sections were incubated with primary antibody for 1 h at 37 °C and 4 °C overnight. After washing with phosphate-buffered saline (PBS), tissues were incubated with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse Ig polymer as a second antibody (Dako, Hamburg, Germany) for 20 min at room temperature, according to the manufacturer’s instructions. Finally, the peroxidase reaction was visualized by incubating the sections with DAB (0.1% phosphate buffer solution, 0.02% diaminobenzidine tetrahydrochloride, and 3% H₂O₂). After rinses in water, the sections were counterstained with hematoxylin and then dehydrated and mounted in resin mount. Negative control slides were processed in parallel using a non-specific immunoglobulin IgG (Sigma Chemical Co, St. Louis, MO, USA) at the same concentration as the primary antibody.

Ji L., Zhang R., Chen J., Xue Q., Moghal N, & Tsao M.S. (2019). PIDD interaction with KEAP1 as a new mutation-independent mechanism to promote NRF2 stabilization and chemoresistance in NSCLC. Scientific Reports, 9, 12437.

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Protein Expression Analysis by Western Blot

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Proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, A, USA). After blocking in 5% nonfat milk for 1 h at room temperature, the membranes were incubated overnight with primary antibodies against ID4 (Cat.# ab49261, Abcam, USA), Notch1 (Cat.# ab52627, Abcam, USA), CBF1 (Cat.# ab180588, Abcam, USA), Hes1 (Cat.# 108937, Abcam, USA), JAG1 (Cat.#ab109536, Abcam, USA), MRP1 (Cat.# ab24102, Abcam, USA), ABCG2 (Cat.# ab24115, Abcam, USA), P-gp (Cat.#ab103477, Abcam, USA) and GAPDH (Cat.#ab8245, Abcam, USA) at 4℃. Then, the membranes were incubated for 1 h at 4℃ with the appropriate HRP-conjugated secondary antibodies (Cat. #7074, Cell Signaling, Beverly, MA, USA). Protein expression levels were detected via enzyme-linked chemiluminescence (Pierce, Rockford, USA).

Zhang X., Gu G., Song L., Wang D., Xu Y., Yang S., Xu B., Cao Z., Liu C., Zhao C., Zong Y., Qin Y, & Xu J. (2020). ID4 Promotes Breast Cancer Chemotherapy Resistance via CBF1-MRP1 Pathway. Journal of Cancer, 11(13), 3846-3857.

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Western Blot Analysis of Key Proteins

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WB experiments were performed as indicated in our former studies 10 . Mouse anti-human STAT3 (ab119352, Abcam, Cambridge, UK), rabbit anti-human P-gp (ab235954, Abcam, Cambridge, UK), mouse anti-human MRP1 (ab24102, Abcam, Cambridge, UK), rabbit anti-human LRP (ab92544, Abcam, Cambridge, UK), and mouse anti-human β-actin (#3700S, CST, MA, USA) were incubated with the membranes overnight at 4°C at dilutions of 1:800, 1:800, 1:800, 1:800, and 1:2000, respectively.

Tian X., Zuo X., Hou M., Li C, & Teng Y. (2021). LncRNA-H19 regulates chemoresistance to carboplatin in epithelial ovarian cancer through microRNA-29b-3p and STAT3. Journal of Cancer, 12(19), 5712-5722.

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Multiplex Protein Detection Procedure

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The following primary antibodies used in this study were purchased from Abcam (Cambridge, UK): anti-MRP1 (ab24102), anti-α-tubulin (ab52866), anti-MRP1 (ab233383), anti-vinculin (ab130007), anti-IDH1 (ab172964), anti-c-Myc tag (ab32), rabbit monoclonal IgG isotype control (ab172730), mouse monoclonal IgG1 isotype control (ab18443), anti-β-catenin (ab231305 and ab35272), anti-GAPDH HRP-conjugated (ab9385), anti-PARP1 (ab32138), anti-cleaved PARP1 (ab110315), anti-AKT1 pSer-473 (ab81283), and anti-AKT1 (ab108202). Antibody binding for multicolor Western blotting was detected using goat anti-rabbit IgG H&L (IRDye® 800CW), goat anti-mouse IgG H&L (IRDye® 680RD), goat anti-mouse IgG H&L (IRDye® 800CW), and goat anti-rabbit IgG H&L (IRDye® 680RD) preadsorbed secondary antibodies (LI-COR Biosciences; 926-32211, 926-68070, 926-32210, and 926-68071). Chemiluminescent detection was achieved using HRP-conjugated goat anti-mouse IgG (H + L) (LI-COR 926-80011; lot no. C80716-02) and HRP-conjugated goat anti-rabbit IgG (H + L) (LI-COR 926-95000; lot no. C90214-01).

Pillai-Kastoori L., Heaton S., Shiflett S.D., Roberts A.C., Solache A, & Schutz-Geschwender A.R. (2019). Antibody validation for Western blot: By the user, for the user. The Journal of Biological Chemistry, 295(4), 926-939.

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Western Blot Analysis of MDR-1 and MRP-1 Proteins

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Hippocampal tissue was properly thawed and washed with phosphate-buffered saline (PBS). Total proteins were extracted ice cold 1X RIPA lysis buffer. One mL of 1X RIPA lysis buffer was added along with 2 μL protease inhibitor (Sigma-Aldrich). The bicinchoninic acid (BCA) method was used to analyze protein concentration using the BCA kit (BioRad). After heating, the samples were resolved on sodium-dodecyl sulphate polyacrylamide gel using 4% stacking and 8% separating gel. Polyvinylidene difluoride (PVDF) membrane was used to blot the resolved proteins. The blocking was performed in the BSA at 25°C. Blotted PVDF membranes were incubated with specific primary antibodies overnight at 4°C, mouse monoclonal MDR-1 at a concentration of 1:200 (Santa-Cruz Biotech., sc-71557), mouse monoclonal MRP-1 at a concentration of 1:50 (Abcam, ab24102), and mouse monoclonal β-actin (an internal control) at a concentration of 1:5000 (Thermo fisher, M5A-15739). After proper washing with tris-buffered saline, 0.1% Tween 20 (TBST) buffer, blotted membranes were incubated with respective secondary antibodies at room temperature–rabbit anti-mouse (1:10,000, Abcam, ab97046). Enhanced chemiluminescence (ECL) kit (BioRad) was used to visualize blotted membranes. To interpret data, ImageJ Plus software was used for the densitometry analysis.

Kaur M., Gupta T., Gupta M., Singla N., Kharbanda P.S., Bansal Y.S., Sahni D., Radotra B.D, & Gupta S.K. (2023). Expressional Study of Permeability Glycoprotein and Multidrug Resistance Protein 1 in Drug-resistant Mesial Temporal Lobe Epilepsy. Basic and Clinical Neuroscience, 14(5), 615-630.

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Assessment of Drug Transporter Expression in H292 Cells

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H292 cells were grown on six-well transmembranes to confluence, rinsed with HBSS, and infected with TIGR4 (MOI of ~10) apically. The cells were washed and biotinylated as previously described (53 (link), 54 (link)). The cells were lysed and passed through 26 1/2-gauge needles, spun, and applied to streptavidin beads (catalog no. 20347; Thermo Fisher) or saved in Tricine sample buffer (recipe from Bio-Rad [catalog no. 161-0739]). Biotinylated-bead samples were incubated at 4°C overnight, washed, incubated at 40°C for 20 min in Tricine sample buffer, then run on 4 to 20% TGX protein gel. The proteins were transferred onto nitrocellulose membranes, blocked, and incubated with antibodies against the following: MRP1 (ab24102; Abcam), MRP2 (ab3373; Abcam), MRP3 (ab3375; Abcam), MRP4 (ab56675; Abcam), MRP5 (ab77369; Abcam), P-glycoprotein (P-gp) (catalog no. 517310; EMD Millipore), and GAPDH (MAB374; EMD Millipore). Horseradish peroxidase (HRP)-conjugated secondary antibodies were used to visualize Western blots.

Zukauskas A., Mrsny R.J., Cortés Barrantes P., Turner J.R., Leong J.M, & McCormick B.A. (2018). Transporters MRP1 and MRP2 Regulate Opposing Inflammatory Signals To Control Transepithelial Neutrophil Migration during Streptococcus pneumoniae Lung Infection. mSphere, 3(4), e00303-18.

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