Total proteins were isolated using tissue lysis with PK buffer (NaH2PO4 0.2 M; Na2HPO4 0.2 M; NaCl 5 M; EDTA) and protease inhibitor buffer (Triton 100X 2%; SDS 0.25%; Leupeptin 0.90%; Pepstatin A 0.90%; PMSF 0.90%), and centrifuged to pellet debris. Protein concentrations were measured by Micro BCA protein assay kit (Thermo Scientific) at 540 nm. Proteins (20 μg) were diluted in NuPAGE® LDS Sample Buffer 4X (Invitrogen) and NuPAGE Reducing Agent 10X (Invitrogen) and electroblotted onto nitrocellulose membranes at 30 Volts for 1 h . Membranes with transferred proteins were incubated with the primary antibody anti-IDH1-R132H (1:100, Dianova) or anti-vinculin (1:5000, Abcam, Cambridge, UK). The primary antibody incubation was followed by incubation with the secondary antibody anti-mouse (1:10000). A chemiluminescence reaction using the ECL (enhanced chemiluminescence) Plus kit (Amersham, GE Healthcare) was detected using film.
Anti idh1 r132h
Manufactured by Dianova
Sourced in Germany
The Anti-IDH1-R132H is a laboratory reagent designed for the detection and analysis of the IDH1-R132H mutation, a common genetic alteration found in certain types of cancer. This product is intended for use in research applications and assay development, providing researchers with a specific tool to identify and study the IDH1-R132H mutation.
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Anti cd31, by BD (1 mentions)
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Anti atrx, by Merck Group (1 mentions)
Anti egfr, by Agilent Technologies (1 mentions)
Anti tp53, by Agilent Technologies (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Gel pro analyzer 4, by Media Cybernetics (1 mentions)
Anti dnmt1, by Abcam (1 mentions)
Anti idh1, by Santa Cruz Biotechnology (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Anti chk2, by Cell Signaling Technology (1 mentions)
8 protocols using anti idh1 r132h
1
Western Blot Analysis of IDH1-R132H Protein
2
Immunohistochemical Analysis of Brain Tumors
3
Protein Expression Analysis of Glioma Biomarkers
4
Western Blot Analysis of Protein Expression
5
Immunohistochemical Detection of IDH1 R132H
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The IHC clone used was anti-IDH1 R132H (Dianova, USA Clone: H09-unconj) in dilution of 1:25. Immunoreactions were scored positive for mutated IDH1 protein when a majority of tumor cells showed strong cytoplasmic positivity with or without nuclear staining.[16 (link)] Normal/residual glial cells and vascular endothelial cells served as an internal negative control.
6
Glioma Protein Profiling by Western Blot
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Glioma cells were treated with lysis buffer and centrifuged at 12,000 ×g. We separated the protein by gel electrophoresis. Then we transferred the protein from the gel to the PVDF membrane. Blocking the membrane in 5% nonfat milk at room temperature for one hour. The membranes were incubated with specific antibodies. All antibodies were diluted at specific concentrations given by manufacturer including anti-IDH1-R132H (Dianova DIA-H09, Hamburg, Germany), anti-ATM (Proteintech, Rosemont, IL), anti–p-ATM, anti-CHK2, anti–p-CHK2, anti-p53, anti–p-p53, anti-H2AX, anti-Bax, anti–Bcl-2 (Cell Signaling Technology, Beverly, MA) and anti-GAPDH (Proteintech). Incubating the membranes with secondary antibody. The membranes were treated with ECL and scanned by Gel Doc 2000 (Bio-Rad, Hercules, CA).
7
Western Blot Analysis of IDH1 Mutations
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Cells were lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (both from Sigma Aldrich). Proteins were separated on 10% SDS–PAGE and transferred to the PVDF membrane (Immobilon—P, Merck Millipore) by electroblotting. Subsequently, membrane was blocked with 5% Skim Milk (Sigma Aldrich) and incubated at 4°C overnight with primary antibodies: anti-IDH1wt (1:1000, D2H1, Cell Signaling), anti-IDH1R132H (1:100, H09, Dianova), anti-Actin (1:4000, MAB1501, Millipore). After washing, membrane was probed with appropriate HRP-conjugated secondary antibodies: anti-rabbit (1:4000, sc-2004, Santa Cruz Biotechnology), anti-mouse (1:4000, sc-2005, Santa Cruz Biotechnology). Bands were visualized with enhanced chemiluminescence (Amersham ECL Prime Western Blotting Detection Reagent, GE Healthcare) on ChemiDoc XRS (Bio-Rad). Densitometric analysis was performed using Image J software.
8
Immunohistochemical Analysis of Tumor Markers
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Immunohistochemistry was performed using protocols described previously by Gurung et al (25) . In brief, the TMA slides were deparaffinised, rehydrated, washed and endogenous peroxidase was blocked using Bond-III 'Dewax Protocol D' following the manufacturer's instructions (Leica Biosystems, Newcastle, UK). Epitope retrieval was achieved using Bond-III 'Protocol H1(30)' (Leica Biosystems). The slides were incubated with antibodies against anti-IDH1-R132H (1:400; Dianova GmbH, Hamburg, Germany), CD133 (1:500; Abcam, Cambridge, UK) or nestin (1:500; Abcam) at room temperature (RT) for 1 h. Antibody binding was detected using diaminobenzidine (DAB) with hematoxylin counterstaining following Bond-max and Bond-x 'IHC protocol F' (Leica Biosystems). The immunostaining results were interpreted independently by two expert pathologists who were blinded to the clinical data. The staining intensity was scored using the following scale of four grades: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. At least 5 areas of each core were viewed and the proportion of cells in each core staining positively was assigned a score (0-100%). A semi-quantitative histopathology (H) score was obtained by multiplying the staining intensity score with the percentage score (0-300). An H-score higher than the median was considered positive.
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