C met

Manufactured by Abcam

Sourced in United States, United Kingdom

C-Met is a recombinant protein that serves as a tool for research and development purposes. It is the extracellular domain of the human c-Met receptor tyrosine kinase, which is involved in cellular processes such as cell growth, survival, and motility. C-Met can be used in various in vitro and in vivo applications to study its role and interactions in biological systems.

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Lab products found in correlation

Bcl 2, by Abcam (3 mentions) Bcl2 associated x (bax), by Abcam (3 mentions) Pvdf membrane, by Merck Group (3 mentions) P akt, by Abcam (2 mentions) Cyclin d1, by Abcam (2 mentions) Bond 3, by Leica Biosystems (2 mentions) Ihc and ish stainer, by Leica Biosystems (2 mentions) Sting, by Cell Signaling Technology (2 mentions) Cleaved caspase 3, by Cell Signaling Technology (2 mentions) P akt, by Cell Signaling Technology (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions) Pc met, by Abcam (2 mentions) N cadherin, by Abcam (2 mentions) Cd133, by Abcam (1 mentions) Dp71 digital imaging system, by Olympus (1 mentions) 3 30 diaminobenzidine, by Vector Laboratories (1 mentions) Braf antibody, by Abcam (1 mentions) Ix71 microscope, by Olympus (1 mentions) P erk1 2, by Abcam (1 mentions) Anti hgf, by Abcam (1 mentions)

23 protocols using c met

Immunohistochemical Evaluation of C-met Expression

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C-met (clone: EP1454, dilution 1:200; Abcam, Cambridge, United Kingdom) was used and pre-heated using heatinduced epitope retrieval (pre-heated in pressure cooker for 5 minutes in citrate buffer, pH 6), which was employed prior to C-met staining. Primary antibodies were then added and slides incubated for 2 hours at 37 C. Slides were then processed on an immunostainer (LabVision Autostainer 360, Fujian, China). The primary antibody was replaced by phosphatebuffered saline as a negative control to assess the specificity of the antibodies. Hematoxylin-counterstained sections were mounted in aqueous mounting medium and observed under a light microscope.
The IHC staining (percentage of stained cells Â staining intensity) for C-met was scored for each case after semiquantitative evaluation by 2 independent pathologists (L.R. and Z.-Y.M.). The percentage of stained cells observed in every 100 positive cells/100 Â 100% was the percentage of positive cells. Staining intensity was negative (0), weak (light The final score of the specimen area was percentage of positive cells Â staining intensity Â 100. The final score for each antibody IHC staining ranged from 0 to 300 points, with a median score as a borderline, and they were assigned to a highexpression group and a low-expression group, and the median value was assigned to the high-expression group.

Mao Z.Y., Zhu G.Q., Ren L., Guo X.C., Su D, & Bai L. (2016). Prognostic Value of C-met Expression in Cholangiocarcinoma. Technology in cancer research & treatment, 15(2).

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Immunohistochemical Profiling of Tumor Samples

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Tumor tissues were excised with proper precautions and fixed in formalin for the preparation of paraffin sections. Paraffin-embedded tumor sections were deparaffinized in xylene and then with 100, 90, 80 and 70% ethanol, followed by phosphate-buffered saline (PBS). Tumor sections were stained with hematoxylin and eosin (H & E) or immunostained overnight at 4^οC with the BRAF antibody (1:300; Abcam), the cMET (1:300; Abcam), the CD133 (1:200; Abcam) antibody, and Ki67 (1:200; Abcam). After washing in PBS, a 1:200 dilution of biotinylated goat anti-rabbit IgG or anti-mouse IgG antibody in a blocking solution was applied to the sections and they were incubated for 30–40 min. Following the PBS treatment, ABC reagent was applied to the sections and they were incubated a further 30 min. Color reaction tests were performed with 3, 30-diaminobenzidine (Vector Laboratories) and the slides were washed twice with PBS. After hematoxylin counterstaining and clearing with a graded ethanol series and xylene, sections were mounted with Canada balsam. Images were photographed using an IX71 microscope (Olympus) equipped with the DP71 digital imaging system (Olympus).

Adhikari M., Kaushik N., Ghimire B., Adhikari B., Baboota S., Al-Khedhairy A.A., Wahab R., Lee S.J., Kaushik N.K, & Choi E.H. (2019). Cold atmospheric plasma and silymarin nanoemulsion synergistically inhibits human melanoma tumorigenesis via targeting HGF/c-MET downstream pathway. Cell Communication and Signaling : CCS, 17, 52.

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Western Blot Analysis of Protein Signaling

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Protein extracts (30 µg per lane) were electrophoresed and then transferred to polyvinylidene fluoride membrane. Immuno-blotting was performed by incubating each membrane with an anti-HGF (dilution: 1∶1000; Abcam, Cambridge, UK), c-MET (dilution: 1∶250; Abcam, Cambridge, UK), AKT (dilution: 1∶1000; Abcam, Cambridge, UK), p-AKT (dilution: 1∶1000; Abcam, Cambridge, UK), ERK1/2 (dilution: 1∶1000; Abcam, Cambridge, UK), p-ERK1/2 (dilution: 1∶1000; Abcam, Cambridge, UK), MMP-2 (dilution: 1∶1000; Abcam, Cambridge, UK), MMP-9 (dilution: 1∶500; Abcam, Cambridge, UK), cyclin D1 (dilution: 1∶250; Abcam, Cambridge, UK) or β-actin overnight at 4°C. After being washed in PBS, each membrane was incubated for 1 h with a secondary antibody conjugated by peroxidase at room temperature. The band was developed by use of enhanced chemi-luminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The density of each band was determined. The results were repeated twice to confirm the reproducibility.

Du T., Ju G., Wu S., Cheng Z., Cheng J., Zou X., Zhang G., Miao S., Liu G, & Zhu Y. (2014). Microvesicles Derived from Human Wharton's Jelly Mesenchymal Stem Cells Promote Human Renal Cancer Cell Growth and Aggressiveness through Induction of Hepatocyte Growth Factor. PLoS ONE, 9(5), e96836.

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Comprehensive Immunohistochemical Analysis of Tumor Microenvironment

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Surgical specimens were available from the 38 patients who underwent surgical resection or exploration. Five-micrometer-thick sections were cut from formalin-fixed, paraffin-embedded blocks for staining with Masson tri-chrome (to assess fibrosis) or antibodies against SMAD4 (Abcam), HGF (Abcam), cMET (Abcam), SDF1α (Bio-Vision), CXCR4 (R&D Systems, Minneapolis, MN), CXCR7 (Abcam), CD31 (Dako), α-SMA (Sigma), and CD68 (Thermo Scientific). Semiquantitative and quantitative analyses for biomarker expression or tumor-associated macrophage number (estimated by positive staining area ratio) was carried out specifically for the intratumoral and stromal (tumor periphery) compartments, and performed by 2 experienced gastrointestinal pathologists. Because, CXCR4 can be expressed either in the cell cytoplasm or the plasma membrane, analysis was performed separately for cytoplasmic and membranous CXCR4 expression. Quantification of tumor blood vessels was separated for immature (non-α-SMA⁺ pericyte-covered) versus more mature (α-SMA⁺ pericyte-covered) vessels.

Hong T.S., Ryan D.P., Borger D.R., Blaszkowsky L.S., Yeap B.Y., Ancukiewicz M., Deshpande V., Shinagare S., Wo J.Y., Boucher Y., Wadlow R.C., Kwak E.L., Allen J.N., Clark J.W., Zhu A.X., Ferrone C.R., Mamon H.J., Adams J., Winrich B., Grillo T., Jain R.K., DeLaney T.F., Castillo C.F, & Duda D.G. (2014). A Phase 1/2 and Biomarker Study of Preoperative Short Course Chemoradiation With Proton Beam Therapy and Capecitabine Followed By Early Surgery for Resectable Pancreatic Ductal Adenocarcinoma. International journal of radiation oncology, biology, physics, 89(4), 830-838.

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EGFR Mutation Analysis in NSCLC

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NSCLC patients with EGFR mutation were identified through the DFCI PROFILE database. Formalin-fixed paraffin embedded (FFPE) with four-micron thick were stained at the division of pathology in Brigham & Women’s Hospital Pathology Core. Staining for the following antibodies was performed on BOND-III, the fully automated IHC and ISH stainer (Leica Biosystems): STING (Cell Signaling Technology, Cat.#13647, dilution 1:50), CD73 (Cell Signaling Technology, Cat.#13160, dilution 1:50), c-MET (Abcam, Cat.#ab243930, dilution 1:100). Poly-HRP IgG reagent from Bond Polymer Refine Detection Kit DC9800 was used to bind rabbit antibody. Expression levels of STING, CD73, and c-MET staining in tumor cells were visually evaluated by a pathologist who was blinded to other data. We used a IHC score system (H-score) that was calculated by multiplying the membranous intensity score in tumor cells (0, absent; 1, weak; 2, moderate; 3, strong) by the percentage of stained cells (0–100%) to yield a value of 0–300m and classified into high versus low expression at the median cut-off point(32 (link)).

Yoshida R., Saigi M., Tani T., Springer B.F., Shibata H., Kitajima S., Mahadevan N.R., Campisi M., Kim W., Kobayashi Y., Thai T.C., Haratani K., Yamamoto Y., Sundararaman S.K., Knelson E.H., Vajdi A., Canadas I., Uppaluri R., Paweletz C.P., Miret J.J., Lizotte P.H., Gokhale P.C., Jänne P.A, & Barbie D.A. (2022). MET-Induced CD73 Restrains STING-Mediated Immunogenicity of EGFR-Mutant Lung Cancer. Cancer Research, 82(21), 4079-4092.

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Immunohistochemical Analysis of Rectal Cancer

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Immunohistochemistry was performed in all but five cases of pre-treatment biopsies, which had insufficient tissue from the rectal biopsy. Resected surgical specimens with residual cancer (TRG2-5) were analyzed as well. Three ìm thick serial paraffin sections of each case were processed by immunohistochemistry using an automated immunostainer (Ventana BenchMark AutoStainer, Ventana Medical Systems, Tucson, AZ, USA) with antibodies against YKL-40 (Quidel Corporation, San Diego, CA, USA, rabbit polyclonal, diluted 1/400) and c-Met (Abcam PLC, Cambridge, UK, rabbit polyclonal, diluted 1:200). The antigen retriewal step was included in the automated programme. A biotin-free, dextran chain-based detection system (EnVysion, Dako, Carpinteria, CA, USA) and diaminobenzidine (Ventana Medical Systems, Tucson, AZ, USA) were used as the chromogen, according to standard protocols. Neoplastic tissues were assembled on tissue microarrays, excluding the primary antibody and IgG-matched serum, and were used as positive and negative controls.

Senetta R., Duregon E., Sonetto C., Spadi R., Mistrangelo M., Racca P., Chiusa L., Munoz F.H., Ricardi U., Arezzo A., Cassenti A., Castellano I., Papotti M., Morino M., Risio M, & Cassoni P. (2015). YKL-40/c-Met Expression in Rectal Cancer Biopsies Predicts Tumor Regression following Neoadjuvant Chemoradiotherapy: A Multi-Institutional Study. PLoS ONE, 10(4), e0123759.

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Western Blot Analysis of Protein Targets

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Extracted protein was used to perform Western blotting as previously described.15 (link) The antibodies, which were diluted in primary antibody diluent, are listed below: KIAA1522 (1/1000, ab122203; Abcam, Cambridge, UK), Bcl-2 (1/1000, ab196495; Abcam, Cambridge, UK), Bax (1/1000, ab53154; Abcam, Cambridge, UK), CyclinD1 (1/1000, ab226977; Abcam, Cambridge, UK), CD44 (1/1000, ab157107; Abcam, Cambridge, UK), c-Met (1/1000, ab74217; Abcam, Cambridge, UK), cleaved caspase-3 (1/1000, 9661; Cell Signaling Technology, Beverly, MA, USA), CDK2 (1/1000, 2546; Cell Signaling Technology, Beverly, MA, USA), CDK4 (1/1000, 12790; Cell Signaling Technology, Beverly, MA, USA), CDK6 (1/1000, 13331; Cell Signaling Technology, Beverly, MA, USA), β-catenin (1/1000, 8480; Cell Signaling Technology, Beverly, MA, USA), and c-Jun (1/1000, 9165; Cell Signaling Technology, Beverly, MA, USA). HRP-conjugated Affinipure goat anti-rabbit (1/5000, A0208; Beyotime, Shanghai, China), or anti-mouse IgG(H+L) (1/5000, A0216; Beyotime, Shanghai, China) was diluted in secondary antibody diluent and used to incubate the membranes.

Jiang S., Zhang Y., Li Q., Qiu L, & Bian B. (2020). KIAA1522 Promotes the Progression of Hepatocellular Carcinoma via the Activation of the Wnt/β-Catenin Signaling Pathway. OncoTargets and therapy, 13, 5657-5668.

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Liver Histology and Immunohistochemistry

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Liver specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Liver histopathologic analysis on mouse lesions was assessed by two experienced liver pathologists (SR and FD) in accordance with the criteria described in detail previously.^{31 (link)} Immunohistochemistry (IHC) was performed as previously described.^{21 (link), 24 (link)} The primary antibodies against c-Met (Abcam, Cambridge, MA, USA; 1:100), p-AKT^S473 (Cell Signaling Technology, Danvers, MA, USA; 1:100), Pten (Cell Signaling Technology; 1:100), fatty acid synthase (FASN; Cell Signaling Technology; 1:150), acetyl-CoA carboxylase (ACC; Cell Signaling Technology; 1:100), p-ERK (Cell Signaling Technology; 1:100), and Ki67 (Thermo Fisher Scientific, Waltham, MA, USA; 1:150) were used in the present investigation.

Xu Z., Hu J., Cao H., Pilo M.G., Cigliano A., Shao Z., Xu M., Ribback S., Dombrowski F., Calvisi D.F, & Chen X. (2018). Loss of Pten synergizes with c-Met to promote hepatocellular carcinoma development via mTORC2 pathway. Experimental & Molecular Medicine, 50(1), e417-.

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Immunofluorescence and Western Blot Analysis

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Phalloidin, 1∶100 was purchased from Molecular Probes. α-Tubulin antibody, 1∶1000, was purchased from Lab Vision. LAMP-1 antibody (H4A3), 1∶50, was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa, USA. The following antibodies were used for Western bloting: pMet, pAkt, pErk1/2, (1∶1000), cleaved-caspase-3 (1–200) (Cell Signaling Technology, Beverly, MA, USA), Rab7 (1∶1000) (Sigma, St Louis, MO, USA), c-Met (tissue samples 1∶500) (Abcam, Cambridge, MA, USA), c-Met (1∶1000) (Invitrogen, Carlsbad, CA, USA), Ki67 (1∶50) (Thermo Fisher Scientific, Rockford, IL). Fluorophore-conjugated secondary antibodies (1∶100) were purchased from Jackson Immunoresearch Laboratories (Westrgrove, PA, USA). IHC secondary antibodies (1∶200) were purchased from Vector Labs (Burlingame, CA, USA). HGF (Calbiochem, San Diego, CA, USA) was used at 33 ng/ml.

Steffan J.J., Dykes S.S., Coleman D.T., Adams L.K., Rogers D., Carroll J.L., Williams B.J, & Cardelli J.A. (2014). Supporting a Role for the GTPase Rab7 in Prostate Cancer Progression. PLoS ONE, 9(2), e87882.

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Protein Expression Analysis in Cultured Cells

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Total proteins from cultured cells were extracted using RIPA Lysis Buffer (Beyotime, Shanghai, China). Approximately equal amounts of proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% non‐fat milk, the membranes were incubated with primary antibodies against c-Met (1:1000, Abcam), Bax (1:1000, Abcam), Bcl-2 (1:1000, Abcam) and GAPDH (1:10,000, Cell Signaling Technology) overnight at 4 °C. The membranes were subsequently exposed to horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. The protein bands of interest were detected using the ECL kit (Millipore), and protein levels were quantified using ImageJ software.

Wang B., Ma L, & Wang J. (2022). LncRNA HOTTIP Knockdown Attenuates Acute Myocardial Infarction via Regulating miR-92a-2/c-Met Axis. Cardiovascular Toxicology, 22(4), 352-364.

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