Ab2074

Manufactured by Abcam

Sourced in United States, United Kingdom

Ab2074 is a laboratory equipment product manufactured by Abcam. It is designed for use in scientific research applications. The core function of this product is to facilitate specific experimental or analytical tasks, however, a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions) Lsm 710 confocal microscope, by Zeiss (3 mentions) Ab33682, by Abcam (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Ab13604, by Abcam (2 mentions) Alexa 488 anti rabbit, by Thermo Fisher Scientific (2 mentions) Total h3 ab1791, by Abcam (2 mentions) Abt 491, by Merck Group (2 mentions) Sc 135886, by Santa Cruz Biotechnology (2 mentions) Sc 9959, by Santa Cruz Biotechnology (2 mentions) Acetyl h3 ab47915, by Abcam (2 mentions) Anti p84 gtx70220, by GeneTex (2 mentions) Anti tubulin, by Santa Cruz Biotechnology (2 mentions) Ab24874, by Abcam (1 mentions) Dylight 488 conjugated goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions) Cy3 conjugated mouse anti α sma, by Merck Group (1 mentions) Anti mmp 9, by Bioss Antibodies (1 mentions) Ab15323, by Abcam (1 mentions) Dabrafenib, by Selleck Chemicals (1 mentions) 3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)

35 protocols using ab2074

Necrosis Assay and Immune Cell Profiling

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For necrosis assay, LLC tumor sections were HE-stained and histopathologically examined to identify necrotic areas in tumor tissues. CD11b+ cells accumulation was immunohistochemically assessed in formalin-fixed, paraffin-embedded 4 μm thick tumor serial sections by using a rabbit anti-mouse CD11b antibody (Bioss, Beijing). Frozen tissue sections were co-immunostained with rat anti-mouse CD11b-FITC (ab24874, Abcam) and rabbit anti-CD31 (SAB1302548, sigma), CXCR4 (ab2074, Abcam), Cy3-conjugated mouse anti-α-SMA (1:400; Sigma) antibodies, anti-iNOS (ab15323, Abcam) or anti-MMP-9 (Bioss, Beijing). Then staining with anti-CD 31 antibody, anti-CXCR4 antibody, anti-iNOS antibody and anti-MMP-9 antibody was followed by staining with a DyLight 488-conjugated goat anti-rabbit secondary antibody (1:200; Jackson ImmunoResearch).

Liu T., Xie C., Ma H., Zhang S., Liang Y., Shi L., Yu D., Feng Y., Zhang T, & Wu G. (2014). Gr-1+CD11b+ cells facilitate Lewis lung cancer recurrence by enhancing neovasculature after local irradiation. Scientific Reports, 4, 4833.

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Dabrafenib Treatment and Protein Expression

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Dabrafenib was purchased from Selleckchem (Houston, TX) and dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) at a final concentration of 1.92 mM. Dabrafenib was stored as stock solutions at -80°C and diluted in CM just before use. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich, dissolved at a concentration of 5 mg/ml in GIBCO™ Phosphate-Buffered Saline (PBS) (Invitrogen, Thermo Fisher Scientific, Waltham, MA) and stored at 4°C. Mouse monoclonal antibodies (mAb) against ADAM9 (sc-377233), and rabbit polyclonal antibody against human β-tubulin (sc-9104) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). mAb against PIK3R2 (#05-217) was purchased from Upstate Biotechnology (Lake Placid, NY). mAb against MMP7 (#111433) was purchased from R&D Systems (Minneapolis, MN). Rabbit polyclonal antibody against CXCR4 (ab2074) was purchased from Abcam (Cambridge, MA). Rabbit polyclonal antibodies against ERK1/2 (#9102), phospho-ERK1/2 (Thr202/Tyr204) (#9101), AKT (#9272) and phospho-AKT (Ser473) (#9271) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Reagents for sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis were all purchased from Bio-Rad Laboratories, Inc. (Hercules, CA).

Caporali S., Amaro A., Levati L., Alvino E., Lacal P.M., Mastroeni S., Ruffini F., Bonmassar L., Antonini Cappellini G.C., Felli N., Carè A., Pfeffer U, & D’Atri S. (2019). miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A. Journal of Experimental & Clinical Cancer Research : CR, 38, 272.

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Multiparametric Flow Cytometry of Vascular and Lymphatic Endothelial Cells

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VECs and AECs were analyzed via flow cytometry for various EC and LEC markers. Cells were harvested using Cell Dissociation Buffer (LifeTechnologies) and fixed in 4% formaldehyde for 1 hr at 4°C. For intracellular staining, cells were additionally treated with ice-cold methanol (−20°C) for 1 hr at 4°C. Primary antibodies for EC markers CD31 (MCA1746 clone LCI-1, AbD serotec, Raleigh, NC), and Flt-1 (sc-31173 clone N-16, Santa Cruz, Dallas, TX), tip-cell/angiogenesis related markers CXCR4 (ab2074, Abcam, Cambridge, MA) and DLL4 (ab7280, Abcam), and lymphatic markers Prox1 (ab33219 clone 5G10, Abcam) and LYVE1 (ab33682, Abcam) were all used at a concentration of 1 μg per million cells in a staining buffer containing 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). Primary antibody incubation occurred at RT for 30 min, with agitation using a vortex at 15 min. Secondary antibodies conjugated with AlexaFluor 488/647 (Invitrogen/Life Technologies and Abcam) were also used at 1 μg per million cells in a staining buffer containing 1 % BSA in PBS. Secondary antibody incubation occurred at RT for 30 min, with agitation using a vortex at 15min. All samples (n=3–6 biological replicates) were run on a BD FACSCantoII (Franklin Lakes, NJ) and analyzed using FlowJo software (FlowJo, Ashland, OR).

Blancas A.A., Balaoing L.R., Acosta F.M, & Grande-Allen K.J. (2016). Identifying Behavioral Phenotypes and Heterogeneity in Heart Valve Surface Endothelium. Cells, tissues, organs, 201(4), 268-276.

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Immunohistochemical Analysis of CXCR4 and CXCL12 in Osteosarcoma

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Hematoxylin-eosin sections from paraffin-embedded tumour samples were reviewed by pathologists and the most representative area was chosen for TMA construction using TMAMaster System (Euroclone SpA, Milano, Italy). CXCR4 protein expression was evaluated by immunohistochemistry in 35 high grade and 13 low grade OS samples. Sections were incubated overnight with rabbit monoclonal anti-CXCR4 antibody (ab2074) (Abcam, Cambridge, GB), and polyclonal anti-CXCL12 (FL-93) (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:1000 in PBS. After, sections were washed and incubated with the streptavidin-biotin peroxidase DAB detection system (Dako, Glosturp, Denmark), according to the manufacturer's protocol.
Sample staining was scored for intensity (0=no visual staining; 1= weak/moderate; 2=strong), and percentage of positive tumour cells (0 ≤ 10%; 1 = =10%–24%; 2 = =25%–49%; 3 ≥ 50%). Cut-off levels, determined by the scores sum, were applied as 0 for negative cases, 1–3 for weak or moderate positivity in less than 50% of tumour cells, and 4–5 for moderate or strong positivity in almost 50% of tumour cells. The latter range was considered protein overexpression.

Pollino S., Palmerini E., Dozza B., Bientinesi E., Piccinni-Leopardi M., Lucarelli E., Righi A., Benassi M.S, & Pazzaglia L. (2019). CXCR4 in human osteosarcoma malignant progression. The response of osteosarcoma cell lines to the fully human CXCR4 antibody MDX1338. Journal of Bone Oncology, 17, 100239.

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Western Blot Analysis of CXCR4 Expression

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Frozen 4T1 tumors and organ tissues were homogenized in NP40 buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40) supplemented with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The lysates were centrifuged at 4 °C and the supernatants were collected as the protein extracts. The protein content of each sample was determined using the Bio-rad protein assay (Bio-rad, Hercules, CA). Eighty μg of total protein from each sample were separated on a 12% SDS-PAGE and then electrophoretically transferred onto nitrocellulose membrane (Bio-rad, Hercules, CA). The membranes were blocked in PBS-T (0.1% Tween-20) containing 5% non-fat milk powder for 1 h to inhibit non-specific binding, and then incubated with rabbit anti-CXCR4 antibodies (dilution 1:1000, ab2074, Abcam, Cambridge, MA) overnight at 4 °C. After washing with PBS-T, the membranes were incubated with horseradish peroxidase-linked antirabbit IgG (dilution 1:5000; GE Healthcare Bio-Sciences, Pittsburgh, PA) for 45 min at RT, followed by chemiluminescent detection with ECL substrate (GE Healthcare Bio-Sciences, Pittsburgh, PA) for 1 min. The images were exposed and captured immediately. For normalization, β-actin was detected as internal standard in parallel blots.

Zhao Y., Detering L., Sultan D., Cooper M.L., You M., Cho S., Meier S.L., Luehmann H., Sun G., Rettig M., Dehdashti F., Wooley K.L., DiPersio J.F, & Liu Y. (2016). Gold Nanoclusters-Doped With 64Cu for CXCR4 Positron Emission Tomography Imaging of Breast Cancer and Metastasis. ACS nano, 10(6), 5959-5970.

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Western Blot Analysis of CXCR4, CXCR7, and Signaling Pathways

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Whole cell extracts were prepared using commercially available RIPA lysis buffer (Thermo, MA, USA). Proteins (40 μg in total) were loaded on a 10% SDS-PAGE gel and transferred to NC membranes (Millipore, MA, USA) by semidry transfer. After blocking for 1 h with 5% (w/v) bovine serum albumin dissolved in 0.1% TBS-T, membranes were incubated overnight at 4 °C with the following primary antibody: anti-CXCR4 (ab2074, Abcam, Cambridge, UK) at 1/50 dilution, anti-CXCR7 (ab117836, Abcam, Cambridge, UK) at 1/50 dilution, anti-phospho-ERK1/2 (#4370, Cell signaling technology, MA, USA) at 1/2000 dilution, anti-ERK1/2 (#9102, Cell signaling technology, MA, USA) at 1/1000 dilution, anti-phospho-Akt (Ser473) (#4060, Cell signaling technology, MA, USA) at 1/2000 dilution, anti-Akt (pan) (#4691, Cell signaling technology, MA, USA) at 1/1000 dilution, anti-Raf-1 (ab18761, Abcam, Cambridge, UK) at 1/50 dilution, anti-GAPDH (#5174, Cell signaling technology, MA, USA) at 1/5000 dilution. After washing in 0.1% TBS-T, membranes were incubated with the appropriate HRP-linked secondary antibody for 1 h at room temperature. Detection of bound antibody was performed using the enhanced chemiluminescence kit (Pierce, Rockford, IL, USA). Bands were visualized using X-ray exposure machine (Genetech, Shanghai, China). All experiments were repeated 3 times (n = 3).

Chen D., Xia Y., Zuo K., Wang Y., Zhang S., Kuang D., Duan Y., Zhao X, & Wang G. (2015). Crosstalk between SDF-1/CXCR4 and SDF-1/CXCR7 in cardiac stem cell migration. Scientific Reports, 5, 16813.

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Immunohistochemical Analysis of Tumor Markers

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The Formalin Fixed Paraffin Embedded (FFPE) sections were de-paraffinized, re-hydrated and antigen retrieval was carried out according to standard protocols. The sections were then incubated with the primary antibodies overnight at 4°C in a humidified chamber. The antibodies used were for CD44 (AM310-5M; Biogenex Life Sciences Pvt. Ltd., Hyderabad, India; Ready-to-use and MA4400; Thermo Fischer Scientific, Rockford, IL, USA; dilution: 1:50), CD31 (endothelial cell clone JC70A (M0823, DAKO, California, USA; dilution: 1:50), CXCR4 (ab2074; Abcam, Cambridge, MA, USA; 1:50) and SDF-1 (97958; Cell signaling technology, Danvers, MA, USA; 1:200). The two CD44 antibodies were evaluated for their comparative staining patterns (Supplementary Figure 1). Sections were then washed twice with 1X-TBST buffer, stained with a secondary detection kit (Real TM EnVision™ Detection system DAKO, Denmark) and counterstained using Mayers-Hematoxylin and mounted using DPX mountant. All the slides were scanned (Aperio ScanScope XT 1509, AT2, Leica biosystems, IL, USA) at 20× magnification, reviewed independently by two observers and images analyzed using the Aperio Imagescope v.11.2.0.780 (Leica biosystems, IL, USA). The reviewers were blinded to the histopathological diagnosis during the entirety of scoring.

Surendran S., Siddappa G., Mohan A., Hicks W J.r., Jayaprakash V., Mimikos C., Mahri M., Almarzouki F., Morrell K., Ravi R., Govindan S., Sushma C.N., Raghavan N., Birur P., Ilayaraja J., Merzianu M., Reid M., Suresh A, & Kuriakose M.A. (2017). Cancer Stem Cell and its Niche in Malignant Progression of Oral Potentially Malignant Disorders. Oral oncology, 75, 140-147.

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Cell Culture Reagents and Antibodies

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RPMI1640, Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT).Trypsin-EDTA (ethylenediaminetetraacetic acid; 0.25%) and antibiotic-antimycotic were obtained from Gibco BRL (Grand Island, NY). Lactacystin and chloroquine were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against CXCR4 (ab2074) was obtained from Abcam (Cambridge, MA). β-Actin was used as a loading control (Cell Signaling, Danvers, MA). CXCL12 was purchased from R&D system (Minneapolis, MN).

Kim B., Yoon J., Yoon S.W, & Park B. (2016). Onbaekwon Suppresses Colon Cancer Cell Invasion by Inhibiting Expression of the CXC Chemokine Receptor 4. Integrative Cancer Therapies, 16(2), 244-251.

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Western Blot Analysis of CXCR4 in BMSCs

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BMSCs cell lysates were prepared in Complete Lysis-M EDTA-free buffer containing protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) and western blots performed as described previously (Periyasamy-Thandavan et al. 2008; Periyasamy-Thandavan et al. 2012). The same amounts of protein (25μg) were loaded for each lane for reducing electrophoresis. The resolved proteins were then electroblotted onto polyvinylidene difluoride membranes. The blots were incubated in a blocking buffer with 5% fat-free milk, and exposed to the primary antibodies overnight at 4 °C, and finally incubated with the horseradish peroxidase-conjugated secondary antibody to reveal antigens using an enhanced chemiluminescence kit from Pierce. Primary antibodies were used for actin (Sigma; A2228) diluted 1:50000 and CXCR4 (Abcam; ab2074) diluted 1:250.

Periyasamy-Thandavan S., Herberg S., Arounleut P., Upadhyay S., Dukes A., Davis C., Johnson M., McGee-Lawrence M., Hamrick M.W., Isales C.M, & Hill W.D. (2015). Caloric Restriction and the Adipokine Leptin alter the SDF-1 signaling axis in Bone Marrow and in Bone Marrow Derived Mesenchymal Stem Cells. Molecular and cellular endocrinology, 410, 64-72.

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Quantitative Western Blot Analysis

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Total protein was isolated from 5×10⁶ cells with 200 ml of ice-cold lysis buffer containing 1% Nonidet P-40 (NP-40), 50 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate, 200 mg/ml phenylmethanesulfonyl fluoride (PMSF), and 50 mg/ml aprotinin. Insoluble materials were removed by centrifugation at 20000 g for 20 min at 4°C. Clarified protein lysates (50 μg) were electrophoretically resolved on a denaturing SDS polyacrylamide gel, and electrotransferred onto nitrocellulose membranes. The membranes were initially blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) for 2 h and then probed with primary antibodies against CXCR4 (ab2074, abcam) and GAPDH (ab9485, abcam) as loading control. Immunodetection was carried out using the ECL Western Blotting Detection Kit (Amersham Corp, UK). Relative protein expression levels were quantified by densitometric measurement of ECL reaction bands and normalized to GAPDH levels.

Lin X.L., Xu Q., Tang L., Sun L., Han T., Wang L.W, & Xiao X.Y. (2017). Regorafenib inhibited gastric cancer cells growth and invasion via CXCR4 activated Wnt pathway. PLoS ONE, 12(5), e0177335.

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