Ab3538p

Manufactured by Merck Group

Sourced in United States

The AB3538P is a laboratory equipment product manufactured by Merck Group. It is a device designed for specific laboratory functions. Due to the need to maintain an unbiased and factual approach, a detailed description of the product's core function cannot be provided without the risk of extrapolation or interpretation. Therefore, a more comprehensive description is not available at this time.

Automatically generated - may contain errors

Lab products found in correlation

Sc 50329, by Santa Cruz Biotechnology (4 mentions) Ab15309, by Abcam (3 mentions) Ecl kit, by Thermo Fisher Scientific (2 mentions) Ab15086, by Abcam (2 mentions) Ab40084, by Abcam (1 mentions) Ventana benchmark xt, by Roche (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) Goat anti rabbit sc 2004, by Santa Cruz Biotechnology (1 mentions) Anti mouse sc2005 igg horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) A11011, by Thermo Fisher Scientific (1 mentions) Alexa fluor plus 680, by Thermo Fisher Scientific (1 mentions) Triton x, by Merck Group (1 mentions) Sc 23900, by Santa Cruz Biotechnology (1 mentions) Sc 9757, by Santa Cruz Biotechnology (1 mentions) Sc 25778, by Santa Cruz Biotechnology (1 mentions) Nxtract, by Merck Group (1 mentions) Horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)

11 protocols using ab3538p

Immunohistochemical Analysis of MCTs and GLUT1

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All applied antibodies had been subjected to validation by the manufacturer for immunohistochemistry (IHC) on paraffin-embedded material, in addition MCT1 and MCT4 was validated by in-house Western blot analysis (Figure 1). All sections were deparaffinised with xylene and rehydrated with ethanol. The 4 µm sections containing tissue cores were subjected to the following antibodies: MCT1 (rabbit polyclonal, AB3538P, Millipore, 1/75), MCT2 (goat polyclonal, ab129290, Abcam, 1∶150), MCT3 (rabbit polyclonal, ab60333, Abcam, 1∶50), MCT4 (rabbit polyclonal, sc-50329, Santa Cruz, 1∶200) and GLUT1 (mouse monoclonal, AB40084, Abcam; 1∶500) [5] (link).
MCT1 and MCT4 were stained using the Ventana Benchmark XT (Ventana Medical Systems Inc.) procedure ultraview DAB. Antigen retrieval was done automatic by CC1 mild (32 min).
For MCT2 and MCT3, antigen retrieval was done manually by placing the specimens in 0.01 M citrate buffer at pH 6.0 and exposed to microwave heating of 20 minutes at 450 W. The primary antibody was visualized by adding a secondary antibody conjugated with Biotin, followed by an Avidin/Biotin/Peroxydase complex (Vectastein ABC Elite kit from Vector Laboratories). Finally, all slides were counterstained with hematoxylin to visualize the nuclei.

Eilertsen M., Andersen S., Al-Saad S., Kiselev Y., Donnem T., Stenvold H., Pettersen I., Al-Shibli K., Richardsen E., Busund L.T, & Bremnes R.M. (2014). Monocarboxylate Transporters 1–4 in NSCLC: MCT1 Is an Independent Prognostic Marker for Survival. PLoS ONE, 9(9), e105038.

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Western Blot Analysis of Metabolic Proteins

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Cells were washed with phosphate-buffered saline (PBS) and lysed with sodium dodecyl sulfate (SDS) lysis buffer [60 mM Tris-HCl, pH 6.8, 1 % SDS in distilled water (DW)] containing protease inhibitor cocktail (Roche, Mannheim, Germany). Equal amounts of protein from each sample were separated by SDS-polyacrylamide gel electrophoresis on 8%–12% gels and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were then incubated with primary antibodies against monocarboyxlate transporters: MCT1 (AB3538P; Millipore), MCT2 and MCT4 (SC50322 and SC50329; Santa Cruz Biotechnology, Santa Cruz, CA, USA), acetyl Co-A synthase 2 (SC85258; Santa Cruz Biotechnology), fatty acid synthase (3189; Cell Signaling Technology, Danvers, MA, USA), and actin (A1978; Sigma-Aldrich, St. Louis, MO, USA). Membranes were washed in PBS and incubated with goat anti-rabbit (sc2004) or anti-mouse (sc2005) IgG horseradish peroxidase (Santa Cruz Biotechnology) as the secondary antibody. Labeled, specific protein bands were visualized using the ECL Kit (Thermo Scientific, Waltham, MA, USA).

Jeon J.Y., Lee M., Whang S.H., Kim J.W., Cho A, & Yun M. (2018). Regulation of Acetate Utilization by Monocarboxylate Transporter 1 (MCT1) in Hepatocellular Carcinoma (HCC). Oncology Research, 26(1), 71-81.

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Neuroblastoma Cell Line Cultivation and Analysis

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The neuroblastoma cell lines were purchased from Shanghai Genechem Co., LTD., and cultured in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C of 5% carbon dioxide. The culture medium was removed once every two days. The neuroblastoma cell lines SK-N-BE2 and IMR-32 are MYCN-amplified, while SK-N-AS, SH-SY5Y and SK-N-SH are MYCN non-amplified cell lines. All the cells utilized in our experiments were confirmed to be free of mycoplasma. 3-BrPA powder was purchased from Sigma Aldrich. Rapamycin powder was purchased from MedChemExpress (MCE). All reagents were dissolved in dimethyl sulfoxide (DMSO, Sigma) and kept at −20°C. Antibodies were used as follows: N-Myc (13987, CST), C-Myc (5605S, CST), MCT1 (AB3538P, Millipore), p-mTOR (Ser2448) (5536S, CST), SQSTM1/p62 (88588S, CST), LC3B (83506S, CST) and β-actin (8457S, CST).

Gan L., Ren Y., Lu J., Ma J., Shen X, & Zhuang Z. (2020). Synergistic Effect of 3-Bromopyruvate in Combination with Rapamycin Impacted Neuroblastoma Metabolism by Inhibiting Autophagy. OncoTargets and therapy, 13, 11125-11137.

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Immunocytochemistry of RKO Cells

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For immunocytochemistry, approximately 2 × 10⁵ of RKO cells were seeded into plastic coverslips and cultured overnight. FAM-RKOpep was diluted in PBS 1X (50 µM) and incubated with adherent RKO cells for 1 h at 4 °C. After rinsing with PBS, the cells were fixed with 4% of PFA for 40 min and then permeabilized with 0.01% Triton-X (Merck) for 5 min. Afterwards, non-specific binding sites were blocked with TBS-T 1X containing 5% BSA for 30 min at 4 °C, and next exposed overnight at 4 °C to the primary antibody (dilution in TBS-T with 5% BSA) MCT1 (1:200 dilution, AB3538P, Merck). After rinsing with PBS 1X, the cells were incubated with anti-rabbit (#A-11011, Thermo Scientific) secondary antibody coupled to Alexa Fluor Plus 680 (1:500 dilution in TBS-T with 5% BSA) for 90 min at room temperature. After being washed with PBS 1X, the cells were stained with Vectashield mounting media containing DAPI. The images were acquired by an Olympus BX51 microscope incorporated with a high-sensitivity camera Olympus DP72 at 100X magnification.

Ferreira D., Silva A.P., Nobrega F.L., Martins I.M., Barbosa-Matos C., Granja S., Martins S.F., Baltazar F, & Rodrigues L.R. (2019). Rational Identification of a Colorectal Cancer Targeting Peptide through Phage Display. Scientific Reports, 9, 3958.

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Plasma Membrane Protein Extraction and Western Blot Analysis

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Total plasma membrane protein was harvested using ultracentrifugation according to Zhang et al. (30 (link)). For tumor samples, tumors were thawed on ice and homogenized in lysis buffer (29 (link)), supplemented with protease inhibitors (20 mL/g tumor). Tumor homogenates were centrifuged at 12,000 rpm for 10 min at 4°C and the resulting supernatants were collected for Western blot analysis. The Western blotting (20 μg total protein per lane) was performed as previously described (29 (link)) using the following antibodies: MCT1 (1:5000; ab3538P, EMD Millipore), MCT4 (1:500; sc50329, Santa Cruz), CD147 (1:100; sc9757, 1:1000; sc9753, Santa Cruz), GLUT1 (1:500; ab40084), the proliferation biomarker Ki67 (1:500; sc23900, Santa Cruz), and GAPDH (1:5000; sc25778, Santa Cruz).

Guan X, & Morris M.E. (2020). In Vitro and In Vivo Efficacy of AZD3965 and Alpha-Cyano-4-Hydroxycinnamic Acid in the Murine 4T1 Breast Tumor Model. The AAPS journal, 22(4), 84.

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Profiling Urothelial Tumor Metabolism

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Freshly resected patient tissue samples were obtained from transurethral resection samples with the approval of the Leeds East Research Ethics Committee and informed patient consent. Specimens were weighed on parafilm, washed in fresh DMEM to remove residual lactate, and then cultured overnight at 37°C in 5% CO₂ in a small volume of fresh DMEM containing 10% FBS. A small volume of culture medium was removed after 4 hours and after overnight culture for determination of lactate levels as described.²² (link) The specimens were histologically graded and staged according to the 1973 World Health Organization recommendations and TNM classification by a trained pathologist. For immunohistochemistry, serial sections of fixed tissue specimens were blocked following antigen retrieval and then stained for target antigens using standard IHC methodology as previously described.³⁷ (link) Antibodies used were: GLUT1 (ab15309, Abcam), LDH‐A (19987‐1‐AP, Proteintech), MCT4 (H90, Santa Cruz Biotechnology), and MCT1 (AB3538P, Merck Millipore). Total RNA from a panel of 263 urothelial tumors was analyzed for mRNA expression levels of LDH‐A, LDH‐B, GLUT1, MCT4, and MCT1 (Supplementary Methods) (Hurst et al, manuscript in revision).

Burns J.E., Hurst C.D., Knowles M.A., Phillips R.M, & Allison S.J. (2021). The Warburg effect as a therapeutic target for bladder cancers and intratumoral heterogeneity in associated molecular targets. Cancer Science, 112(9), 3822-3834.

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Extraction and Quantification of Cellular Proteins

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Nuclear and cytosolic proteins were extracted from MLS-1765 cells, with a nuclear extraction kit (NXTRACT, Sigma-Aldrich) according to the manufacturer’s instructions.
Protein quantification was performed according to the Bio-Rad Dc Protein Assay (500–0113, Bio Rad) and Western blot was performed as previously described [16 (link)]. Briefly, after incubation with the primary polyclonal antibodies rabbit anti-MCT1 (1:200 dilution; AB3538P; Chemicon International), rabbit anti-Histone H3 (1:1000 dilution; ab1791, Abcam), membranes were incubated with the respective secondary antibody coupled to horseradish peroxidase (SantaCruz Biotechnology). The bound antibodies were visualized by chemiluminescence (Supersignal West Femto kit; Pierce).

Pinheiro C., Penna V., Morais-Santos F., Abrahão-Machado L.F., Ribeiro G., Curcelli E.C., Olivieri M.V., Morini S., Valença I., Ribeiro D., Schmitt F.C., Reis R.M, & Baltazar F. (2014). Characterization of monocarboxylate transporters (MCTs) expression in soft tissue sarcomas: distinct prognostic impact of MCT1 sub-cellular localization. Journal of Translational Medicine, 12, 118.

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Immunohistochemical Analysis of MCT and CD147

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MCT1 immunohistochemistry was performed according to the avidin-biotin-peroxidase complex method (R.T.U. VECTASTAIN Elite ABC Kit (Universal), Vector Laboratories, Burlingame, CA), with primary antibody for MCT1 (AB3538P, Chemicon International, Temecula, CA) diluted 1:200, as previously described [17 (link)]. Immunohistochemistry for MCT2, MCT4 and CD147 was performed according to the streptavidin-biotin-peroxidase complex principle (Ultravision Detection System Anti-polyvalent, HRP, Lab Vision Corporation, Fremont, CA), using primary antibodies raised against MCT2 (sc-50322, Santa Cruz Biotechnology, Santa Cruz, CA), MCT4 (sc-50329, Santa Cruz Biotechnology, Santa Cruz, CA), and CD147 (18–7344, ZYMED Laboratories Inc., South San Francisco, CA), diluted 1:100, 1:500 and 1:750, respectively, as previously described [28 (link)]. Negative controls were performed by the use of appropriate serum controls for the primary antibodies (N1699, Dako, Carpinteria, CA). Colon carcinoma tissue was used as positive control for MCT1, MCT4 and CD147 while kidney was used for MCT2. Tissue sections were counterstained with hematoxylin and permanently mounted.

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Immunofluorescence and Western Blot Assays

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The following antibodies and conditions were used for immunofluorescence (IF) and Western blot (WB) assays: MCT1 ((AB3538P, Chemicon International, MERCK, Germany (IF); 1:200), (H-1, sc-365501, Santa Cruz Biotechnology, USA (WB); 1:500)); MCT4 (H-90, sc-50329, Santa Cruz Biotechnology, USA; 1:500); hypoxia-inducible factor 1α (HIF-1α) (610958, BD Biosciences, Germany; 1:100 dilution (IF); 1:500 dilution (WB)), carbonic anhydrase IX (CAIX) (Abcam, UK, 1:2000), and hexokinase II (HKII) (Abcam, UK, 1:750).

Miranda-Gonçalves V., Gonçalves C.S., Granja S., Vieira de Castro J., Reis R.M., Costa B.M, & Baltazar F. (2021). MCT1 Is a New Prognostic Biomarker and Its Therapeutic Inhibition Boosts Response to Temozolomide in Human Glioblastoma. Cancers, 13(14), 3468.

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Comprehensive Immunofluorescence and Western Blot Protocol

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For immunofluorescence (IF) and Western blot (WB) assays, we used the following antibodies and dilutions: VEGF [(1:100 dilution (IF), 1:500 dilution (WB), RM 9128-S0; NeoMarkers)]; Bevacizumab (1:100 dilution (IF), Genentech); MCT1 [(1:200 dilution, AB3538P; Chemicon International (IF)), (1:500 dilution; H-1, sc-365501; Santa Cruz Biotechnology (WB))]; MCT4 (1:500 dilution; H-90; sc-50329; Santa Cruz Biotechnology); CD147 (1:500 dilution, 1.BB.218, sc-71038; Santa Cruz Biotechnology); HKII (1:4000 dilution, ab104836, Abcam); GLUT-1 (1:500 dilution, ab15309, Abcam); HIF-1α [(1:100 dilution (IF); 1:500 dilution (WB), 610958, BD Biosciences), LDHA (1:1000 dilution, E9, sc-137243, Santa Cruz Biotechnology), CAIX (1:2000 dilution, ab15086, Abcam); LC3A/B (1:1000 dilution, 4108, Cell Signaling Technology); p62 (1:1000 dilution, sc-28359, Santa Cruz Biotechnology); IRF1 (1:1000 dilution, sc-135952, Santa Cruz Biotechnology); α-Tubulin (1:5000 dilution, B-5-1-2, sc-2394, Santa Cruz Biotechnology) and GAPDH (1:1000 dilution, sc-69778, Santa Cruz Biotechnology).

Miranda-Gonçalves V., Cardoso-Carneiro D., Valbom I., Cury F.P., Silva V.A., Granja S., Reis R.M., Baltazar F, & Martinho O. (2017). Metabolic alterations underlying Bevacizumab therapy in glioblastoma cells. Oncotarget, 8(61), 103657-103670.

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