Cellular protein was extracted with a cell lysate. Samples were separated using 12% SDS-PAGE. After transferring to nitrocellulose membrane (Millipore, Burlington, MA), the membrane was blocked with TBST containing 5% skim milk powder for 2 h. After washing with PBS for 3 times, membrane was incubated with primary antibodies (1:1000) overnight at 4 °C. After washing with PBS for 3 times, membrane was incubated with secondary antibodies (1:2000) for 4 h at 25 °C. Protein bands were analysed through Image J software. The antibodies used in this study were listed as follows: rabbit monoclonal to H2A histone family member X (H2AX, Abcam, Cambridge, UK; #ab229914), rabbit polyclonal to transient receptor potential melastatin 2 (TRPM2, Abcam, #ab11168), rabbit polyclonal to GAPDH (Abcam, #ab9485) and goat anti-rabbit IgG antibody (ab205718).
Ab11168
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab11168 is a mouse monoclonal antibody that recognizes the human CD45 antigen. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of all nucleated hematopoietic cells. This antibody can be used for the identification and enumeration of CD45-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab229914, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab195352, by Abcam (1 mentions)
Ab52939, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Ab173539, by Abcam (1 mentions)
Ab229136, by Abcam (1 mentions)
Ab18255, by Abcam (1 mentions)
Ab9482, by Abcam (1 mentions)
Fv1200 confocal microscope, by Olympus (1 mentions)
Goat anti rabbit igg hrp, by Thermo Fisher Scientific (1 mentions)
Neutravidin beads, by Thermo Fisher Scientific (1 mentions)
Sulfo nhs ss biotin, by Thermo Fisher Scientific (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti pakt 66444 1 ig, by Proteintech (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti p gsk 3β, by Cell Signaling Technology (1 mentions)
6 protocols using ab11168
1
Immunoblot Analysis of Cellular Proteins
2
Protein Expression Analysis in PA Patients
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Part of the tissues of TT and TP in PA patients was homogenized in lysis buffer containing protease and phosphatase inhibitor (Sigma-Aldrich). Protein samples were separated on SDS-PAGE gel and transferred to nitrocellulose membrane. After blocking, incubate with TRPM2, GAPDH, Ras, Raf-1, PSPH, OASL, PKC, METTL3, HIST1H2AE, cPLA, and AQR antibodies (abcam, ab11168, ab9482, ab52939, ab173539, ab211418, ab229136, ab205791, ab195352, ab18255, ab53421, ab205303) at 4 °C overnight. After incubating with the secondary antibody at 20 °C for 1 h, the membrane was washed 3 times. The immune response zone is detected by the ECL detection system. The protein bands were quantified using Image J software (NIH, Bethesda, MD, USA).
3
TRPM2 and Lysosome Colocalization
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Briefly, the mASMCs grown on coverslips were fixed by 4% paraformaldehyde in PBS, permeabilized with 0.4% Triton X-100, following by incubation with primary antibodies in blocking solution at 4 °C overnight. After three subsequent washes at RT with PBS, the cells were then incubated with fluorochrome-conjugated secondary antibody (1:1000 dilution) for 1 h at room temperature in the dark. TRPM2 (Abcam, ab11168, 1:1000 dilution) and Lamp-1 (Abcam; 1:500 dilution) antibodies were used for TRPM2 and lysosome colocalization detection, respectively. The fluorescence images were acquired with Olympus FV1200 confocal microscope. The colocalization of TRPM2 and Lamp-1 was quantified by Pearson’s correlation coefficient (PCC), which was performed readily using FV1200 software.
4
Biotinylation Assay and Western Blot
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Biotinylation assays and Western blot were conducted as previously described (Lu et al., 2015 (link); Zhang et al., 2015 (link)). In brief, after transfection for 24–36 h, HEK293 cells were rinsed with ice-cold PBS. The cells were subsequently incubated with a fresh preparation of 0.5 mg/ml Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific) dissolved in PBS with Tween-20 for 30 min. Subsequently, unreacted biotin was quenched with PBS containing 100 mM glycine. The cells were lysed with RIPA buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, and 1% sodium deoxycholate, pH 7.4) and subjected to centrifugation at 12,000 g for 15 min. The resulting supernatant was incubated with 40 µl of a 50% slurry of NeutrAvidin beads (Thermo Fisher Scientific) for 2 h at 4°C with continuous rotation. After several washes with RIPA buffer, the biotinylated proteins were eluted from the NeutrAvidin beads with 60 µl of 2× SDS sample buffer. The primary antibody used was rabbit anti–TRPM2 (Ab11168; Abcam), and the secondary antibody was goat anti–rabbit IgG-HRP (1:10,000; 31420; Thermo Fisher Scientific).
5
TRPM2 Overexpression and Signaling
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pcDNA3.1-TRPM2-S-Flag was synthesized by Youbio Co., Ltd. The following antibodies were used in this study: anti-TRPM2-L rabbit polyclonal antibody (ab11168; Abcam); anti-TRPM2-S & -L rabbit polyclonal antibody (bs-2888R; Bioss); anti-Flag (K200001M; Solarvbio); anti-PTEN (60300-1-Ig; Proteintech); anti-pAKT (66444-1-Ig; Proteintech); anti-pGSK3β (#9327S; Cell signaling technology); anti-β-Caternin (#8480S; Cell signaling technology); anti-NF-Kβ (#3033S; Cell signaling technology); anti-pERK (#4370S; Cell signaling technology); anti-Bcl-2 (#3498S; Cell signaling technology); anti-cleaved caspase3 (#9664S; Cell signaling technology); anti-GAPDH mouse monoclonal antibody (60004-1-Ig; Proteintech).
6
Citronellal's Antioxidant and Mitochondrial Effects
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Citronellal (CT, Beijing Yuke Biotechnology Co., Ltd) was purchased form Beijing, China. streptozotocin (STZ), metformin (DMBG), acetylcholine (Ach) and sodium nitroprusside (SNP) were purchased from Signal-Aldrich, St. Louis, MO, USA. The kits for MDA (A003-1-2), NO (C009-2-1), ROS (E004-1-1), and SOD (A001-3-2) were obtained from Nanjing Jiancheng, Nanjing, China. Dihydroethidium (DHE, S0063, Beyotime), ROS assay kit (ROS, S0033S, Beyotime), and JC-1-mitochondrial membrane potential assay kit (C2003S, Beyotime) were obtained from Beyotime, shanghai, China. The anti-NOX2 antibody (ab129068, Abcam), anti-NOX4 antibody (ab154244, Abcam), anti-DRP1 antibody (ab56788, Abcam), anti-TRPM2 antibody (ab11168, Abcam) and anti-VDAC (ab154856, Abcam) were obtained from Abcam, Cambridge, MA, USA. The anti-NHE1 antibody (bs-0505R, Bioss) and anti-α-SMA antibody (bsm-52396R, Bioss) were obtained from Bioss, Beijing, China. The anti-GAPDH antibody (AB0036, Abways) was acquired from Shanghai, China.
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