Clone sp44

Manufactured by Roche

Sourced in United Kingdom

The Clone SP44 is a laboratory instrument designed for DNA cloning and sequencing applications. It functions as a thermal cycler, providing precise temperature control and cycling capabilities required for various molecular biology techniques.

Automatically generated - may contain errors

Lab products found in correlation

Clone ep105by, by Abcam (2 mentions) Clone d13.14.4e, by Cell Signaling Technology (2 mentions) Discovery xt processor, by Roche (2 mentions) Clone 44, by Cell Marque (2 mentions) Clone 138g6, by Cell Signaling Technology (2 mentions) Clone 4b5, by Roche (2 mentions) Ep38y, by Abcam (2 mentions) Clone m1, by Roche (2 mentions) Clone mrq28, by Cell Marque (2 mentions) Benchmark autostainer, by Roche (1 mentions) Ventana benchmark ultra, by Roche (1 mentions) X tra adhesive slides, by Leica (1 mentions) Clone g219 1129, by Roche (1 mentions) G219 1129, by Roche (1 mentions) Ish iview kit, by Roche (1 mentions)

8 protocols using clone sp44

Evaluating MET Amplification in Samples

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MET amplification was determined by MET FISH (Roche/Ventana) and NGS. FISH assays were performed with laboratory-developed reagents as previously described^{10 (link)}. Amplification by FISH was considered present when the MET/CEP7 ratio was > 2.2.
IHC for MET (clone SP44, Roche/Ventana) was independently validated at each site. MET IHC was defined as positive if the sample had an H-score ≥ 200, following a previously established method^{11 (link)}. Pathologist training and interlaboratory proficiency testing were used for IHC scoring (Supplemental Methods).

Guo R., Berry L.D., Aisner D.L., Sheren J., Boyle T., Bunn PA J.r., Johnson B.E., Kwiatkowski D.J., Drilon A., Sholl L.M, & Kris M.G. (2019). MET IHC is a Poor Screen for MET Amplification or MET exon 14 mutations in Lung Adenocarcinomas: Data from a Tri-Institutional Cohort of the Lung Cancer Mutation Consortium. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer, 14(9), 1666-1671.

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Immunohistochemical Validation of C-Met, TrkA, and pERK

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C-Met immunohistochemistry was performed with clone SP44 (Ventana Medical Systems) at a concentration of 9.75 μg/ml and ready to use dilution. Trk A immunohistochemistry was performed with clone EP105BY (Abcam) at a concentration of 0.643 mg/ml and 1:750 dilution. Both C-Met and Trk A immunohistochemistry are clinical validated and were performed in a CLIA accredited laboratory. pERK immunohistochemistry was performed with clone D13.14.4E (Cell Signaling Technology) at a concentration of 1 μg/ml by the Molecular Cytology Core Facility at Memorial Sloan Kettering Cancer Center using Discovery XT processor (Ventana Medical Systems).

Cocco E., Schram A.M., Kulick A., Misale S., Won H.H., Yaeger R., Razavi P., Ptashkin R., Hechtman J.F., Toska E., Cownie J., Somwar R., Shifman S., Mattar M., Selçuklu S.D., Samoila A., Guzman S., Tuch B.B., Ebata K., de Stanchina E., Nagy R.J., Lanman R.B., Houck-Loomis B., Patel J.A., Berger M.F., Ladanyi M., Hyman D.M., Drilon A, & Scaltriti M. (2019). Resistance to TRK inhibition mediated by convergent MAP kinase pathway activation. Nature medicine, 25(9), 1422-1427.

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Comprehensive Genomic Profiling of Lung Cancer

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DNA extraction and mutation analysis of prestudy tumor samples were performed in a central laboratory (Theragen Etex, Suwon, Korea). Mutational status was analyzed by Ion Torrent deep-amplicon sequencing using an Ion Torrent AmpliSeq Cancer Hotspot customized lung cancer panel (Thermo Fisher Scientific Inc., Waltham, MA). Fluorescent in situ hybridization (FISH) of MET (c-MET Probe KBI-10719, Kreatech, LG Amsterdam, Netherlands), EGFR, and HER2 was performed on available tumor tissues using the standard protocol [7 (link),12 (link)], and MET expression status was also analyzed using immunohistochemistry (IHC) (clone SP44, Ventana, Tucson, AZ). Tumors with a staining-intensity score of 3 and with more than 50% positive nuclei were considered to have MET overexpression.

Han J.Y., Lee K.H., Kim S.W., Min Y.J., Cho E., Lee Y., Lee S.H., Kim H.Y., Lee G.K., Nam B.H., Han H., Jung J, & Lee J.S. (2016). A Phase II Study of Poziotinib in Patients with Epidermal Growth Factor Receptor (EGFR)-Mutant Lung Adenocarcinoma Who Have Acquired Resistance to EGFR–Tyrosine Kinase Inhibitors. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 49(1), 10-19.

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C-Met Immunohistochemical Analysis of FFPE Samples

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Archival FFPE material from all consenting study participants was requested for immunohistochemical analysis (IHC) of c-Met using monoclonal antibodies against c-Met (clone SP44; Ventana, Tucson, AZ, USA). The staining was conducted using the Ventana BenchMark Autostainer as per the company's recommended protocol (SP44) as reported previously [14 (link)]. Expression levels of c-Met were assessed semi-quantitatively using the standard H score ≥200.

Chia S.K., Ellard S.L., Mates M., Welch S., Mihalcioiu C., Miller WH J.r., Gelmon K., Lohrisch C., Kumar V., Taylor S., Hagerman L., Goodwin R., Wang T., Sakashita S., Tsao M.S., Eisenhauer E, & Bradbury P. (2017). A phase-I study of lapatinib in combination with foretinib, a c-MET, AXL and vascular endothelial growth factor receptor inhibitor, in human epidermal growth factor receptor 2 (HER-2)-positive metastatic breast cancer. Breast Cancer Research : BCR, 19, 54.

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Immunohistochemical Validation of C-Met, TrkA, and pERK

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Immunohistochemical Evaluation of Protein Expression

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MET IHC was performed on 1–2 µm thick FFPE slides cut and mounted on X-tra Adhesive Slides (Leica, Wetzlar, Germany) using a Ventana Benchmark Ultra automated immunostainer and the clone SP44 (Ventana Medical Systems, Tucson, AZ) as previously described [13] (link). Each sample was assessed using the following scoring criteria for staining intensity: 0 = no staining or <50% of tumor cells with any intensity; 1+ = ≥50% of tumor cells with weak staining but <50% with moderate or higher intensity, 2+ = ≥50% of tumor cells with moderate or higher staining but <50% with strong intensity and 3+ = ≥50% of tumor cells were stained with strong intensity. Score 2+ and 3+ were defined as positive and score 0 and 1+ as negative.

Heydt C., Becher A.K., Wagener-Ryczek S., Ball M., Schultheis A.M., Schallenberg S., Rüsseler V., Büttner R, & Merkelbach-Bruse S. (2019). Comparison of in situ and extraction-based methods for the detection of MET amplifications in solid tumors. Computational and Structural Biotechnology Journal, 17, 1339-1347.

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Immunohistochemical Staining Protocol

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IHC was performed with a Ventana XT automated staining instrument. Antibodies recognizing the following targets were used: MutL homolog 1 (MLH1, ready to use, clone M1, Roche, Basel, Schweiz), MutS protein homolog 2 (MSH2, ready to use, clone G219-1129, Roche), MutS homolog 6 (MSH6, 1:100, clone 44, Cell Marque, Rocklin, CA), postmeiotic segregation increased 2 (PMS2, 1:40, clone MRQ28, Cell Marque), HER2 (ready to use, clone 4B5, Roche), EGFR (1:100, EP38Y, Abcam, Cambridge, UK), c-MET (ready to use, clone SP44, Roche), PTEN (1:100, clone 138G6, Cell signaling, Danvers, MA), and p53 (1:300, DO7, Novocastra, Newcastle, UK). Sections were deparaffinized using EZ Prep solution (Ventana Corporation, Tucson, AZ). CC1 standard (pH 8.4 buffer containing Tris/borate/EDTA) was used for antigen retrieval and blocked with inhibitor D (3% H₂O₂) for 4 min at 37°C. Slides were incubated with primary antibody for 40 min at 37°C, followed by a universal secondary antibody for 20 min at 37°C. Slides were incubated in streptavidin-horseradish peroxidase (SA-HRP) D for 16 min at 37°C, after which the substrate, 3,3′-diaminobenzidine tetrahydrochloride (DAB) H₂O₂, was added for 8 min, followed by hematoxylin and bluing reagent counterstaining at 37°C.

Kim H.S., Shin S.J., Beom S.H., Jung M., Choi Y.Y., Son T., Kim H.I., Cheong J.H., Hyung W.J., Noh S.H., Chung H., Park J.C., Shin S.K., Lee S.K., Lee Y.C., Koom W.S., Lim J.S., Chung H.C., Rha S.Y, & Kim H. (2016). Comprehensive expression profiles of gastric cancer molecular subtypes by immunohistochemistry: implications for individualized therapy. Oncotarget, 7(28), 44608-44620.

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Immunohistochemistry Profiling of Biomarkers

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IHC was performed on a Ventana XT automated staining instrument (Ventana Medical Systems, Tucson, AZ, USA). The following target-specific antibodies were used according to the manufacturer's instructions and a previous study [20 (link)]: MutL homolog 1 (MLH1, ready to use, clone M1, Roche, Basel, Switzerland), MutS protein homolog 2 (MSH2, ready to use, clone G219-1129, Roche), MutS homolog 6 (MSH6, 1:100, clone 44, Cell Marque, Rocklin, CA, USA), postmeiotic segregation increased 2 (PMS2, 1:40, clone MRQ28, Cell Marque), ERBB2 (ready to use, clone 4B5, Roche), EGFR (1:100, EP38Y, Abcam, Cambridge, UK), c-MET (ready to use, clone SP44, Roche), PTEN (1:100, clone 138G6, Cell Signaling, Danvers, MA, USA), and p53 (1:300, DO7, Novocastra, Newcastle, UK). Epstein-Barr virus-encoded small RNAs (EBER) in situ hybridization (ISH) was performed using a Ventana Benchmark ISH system and ISH iView kit (Ventana Medical Systems, Tucson, AZ, USA).

Kim H.S., Lee H., Shin S.J., Beom S.H., Jung M., Bae S., Lee E.Y., Park K.H., Choi Y.Y., Son T., Kim H.I., Cheong J.H., Hyung W.J., Park J.C., Shin S.K., Lee S.K., Lee Y.C., Koom W.S., Lim J.S., Chung H.C., Noh S.H., Rha S.Y., Kim H, & Paik S. (2017). Complementary utility of targeted next-generation sequencing and immunohistochemistry panels as a screening platform to select targeted therapy for advanced gastric cancer. Oncotarget, 8(24), 38389-38398.

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