Anti-MCM9 antibody was generated by immunizing rabbits with GST-MCM9 (residues 936–1135) in HuaBio. The following antibodies were purchased: anti-MCM8 (Proteintech, 1645-1-AP), anti-HORMAD1 (Proteintech, 13917-1-AP), anti-MSH2 (Proteintech, 15520-1-AP), anti-MLH1 (Santa Cruz, sc-271978), anti-UHRF1 (Santa Cruz, sc-373750), anti-α-tubulin (Genscript, A01410-100), anti-FLAG (Sigma, F1804), anti-HA (Sangong, D199961), anti-γH2AX (Abcam, ab81299), and anti-RAD51 (Santa Cruz, sc-8349).
Anti γh2ax
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Anti-γH2AX is a primary antibody used to detect phosphorylated histone H2AX, a marker of DNA double-strand breaks. It is commonly used in the study of DNA damage and repair processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag, by Merck Group (6 mentions)
Anti rad51, by Abcam (6 mentions)
Anti β actin, by Merck Group (5 mentions)
Anti β actin, by Abcam (4 mentions)
Anti igf2bp1, by Santa Cruz Biotechnology (4 mentions)
Anti actin, by Merck Group (3 mentions)
Anti p21, by Santa Cruz Biotechnology (3 mentions)
Anti parp, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Proteintech (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Anti chk1, by Cell Signaling Technology (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Anti cleaved caspase 3, by Abcam (3 mentions)
Anti ki67, by Abcam (3 mentions)
Anti rad51, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Anti p53, by Santa Cruz Biotechnology (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Horseradish peroxidase, by GE Healthcare (2 mentions)
Anti h2ax, by Abcam (2 mentions)
98 protocols using anti γh2ax
1
Antibody Generation and Characterization
2
Doxycycline-Induced Apoptosis Analysis
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Cells were harvested 12 h after doxycycline treatment and lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (Quartett) and a phosphatase inhibitor cocktail (Sigma). For BIM detection, the pan-caspase inhibitor Z-VAD-FMK (MedChemExpress) was added 30 min prior to the addition of doxycycline. The cell lysates were then incubated for 1 h on ice and centrifuged at 15,000g for 30 min at 4 °C. Total protein concentrations for the whole-cell lysates were measured using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of the lysates were resolved on SDS-PAGE gels and transferred to PVDF membranes. These were then incubated with primary antibody overnight at 4 °C before being washed three times with TBS-T buffer and incubated with a secondary antibody for 1 h at room temperature. Chemiluminescence was generated with ECL Western Blotting Substrate (Thermo Fisher Scientific) and detected using a ChemiDoc MP (Bio-Rad). All images were analyzed using Image Lab (Bio-Rad). The antibodies used for the western blots were: anti-FLAG (Sigma, F1804), anti-BCL-xL (Abcam, ab32370), anti-MCL-1 (Abcam, ab32087), anti-γ-H2A.X (Abcam, ab81299), anti-PARP (Abcam, 191217), anti-β-actin (Abcam, ab6276), HRP-conjugated anti-rabbit IgG (Abcam, ab205718), and HRP-conjugated anti-mouse IgG (Abcam, ab6728).
3
Investigating Apoptosis Pathways in Lymphoma
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OCI-LY3 and OCI-LY10 cells were kindly provided by professor Wenbin Qian of Zhejiang University (Zhejiang, China). Z-VAD-FMK and SB203580 were obtained from Selleck Chemicals (Houston, TX). CCK-8 Assay kit was purchased from Meilunbio (Dalian, China). N-acetylcysteine (NAC) and glutathione (GSH) were obtained from Abcam (Cambridge, MA). The Cell Apoptosis detection kit and Cell Cycle Staining Kit were purchased from MultiSciences (Hangzhou, China). Primary antibodies, including anti-cleaved-PARP (ab191217), anti-cleaved-Caspase 3(ab32042), anti-Bax (ab32503), anti-Bad(ab32445), anti-Bcl2(ab32124), anti-β-Actin(ab179467), anti-p21(ab109520), anti-p27(ab32034), anti-p53 (ab179477), anti-p38(ab170099), anti-chk1(ab40866), anti-p-chk2, and anti-γ-H2AX, were purchased from abcam (Cambridge, MA). Antibodies including anti-p-p38(9215S), anti-chk2(3440S), anti-p-chk1(2348P) were purchased from Cell Signaling Technology (Massachusetts, USA). Secondary antibodies, including goat anti-rabbit and goat anti-mouse IgG-HRP were obtained from Beyotome (Shanghai, China).
4
Protein Expression and Localization Assays
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Protein expression levels and intracellular translocations were assessed using cytosolic and nucleic buffers, and standard protocols for western blotting as detailed previously [19 (link)]. The antibodies used were: anti-acetylated histone H3 (Upstate, Overijse, Belgium), anti-actin, anti-Erk (both Sigma-Aldrich), anti-Bax, anti-Bcl-2 (both Dako Cytomation, Heverlee, Belgium), anti-Bid, anti-cytochrome c (Becton Dickinson), anti-BclxL, anti-phospho-Erk, anti-caspase 8, anti-caspase 9 (Cell Signaling, Leiden, the Netherlands), anti-γH2AX and anti-VDAC1 (Abcam, Cambridge, UK).
5
Comprehensive Western Blot Protocol
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Western blot was performed as previously described 44 (link). The antibodies included anti-Oct4 (Bioss, Beijing, China, bs-0830R, 1:1000), anti-Nanog (Proteintech, Wuhan, China, 14295-1-AP, 1:1000), anti-CD44 (Proteintech, 15675-1-AP, 1:1000), anti-Sox2 (Proteintech, 11064-1-AP, 1:1000), anti-γ-H2AX (Abcam, Cambridge, UK, ab26350, 1:1000), anti-DLG2 (Affinity Biosciences, Suzhou, China, DF3995, 1:1000), anti-p-Yap1 (CST, Boston MA, USA, 4911S, 1:1000), anti-Yap1 (Proteintech, 13584-1-AP, 1:1000), anti-Bax (CST, B8429, 1:1000), anti-Bcl2 (Proteintech, 60178-1-Ig, 1:1000), anti-β-tubulin (Sigma-Aldrich, T5201, 1:1000), anti-Lamin B1 (Santa, Dallas TX, USA, sc-365962, 1:1000), anti-TEAD1 (ABclonal, Wuhan, China, A13366, 1:1000), anti-β-actin (Santa, sc-8432, 1:4000), CD63 (Proteintech, 25682-1-AP, 1:1000), and TSG101 (Proteintech, 28283-1-AP, 1:1000).
6
Protein Expression Analysis in GC-1 Cells
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GC-1 cells were lysed in RIPA buffer (Millipore, USA) with 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml CompleteTM EDTA free protease inhibitor cocktail (Roche, USA) and phosphatase inhibitors (5 mM sodium orthovanadate). Protein lysates were loaded on to SDS-PAGE gels, transferred to nitrocellulose membranes (Amersham Biosciences, Germany), immunoblotted with antibodies and visualized using enhanced chemiluminescence substrate (Thermo Fisher Scientific, USA). Protein levels of p53, p21 and γ-H2AX were normalized to GAPDH. The primary antibodies in this study were the following: anti-GAPDH (Cell Signaling Technology, USA), anti-p53 (Santa Cruz, USA), anti-γ-H2AX (Abcam, USA) and anti-p21 (Santa Cruz).
7
Immunoblot Analysis of Cellular Proteins
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Immunoblots were carried out according to standard methods [77 (link)] using rabbit antibody anti-PARP (Cell Signalling Technology Inc., Danvers, MA, USA Upstate, Lake Placid, NY, USA), anti-H2AX, anti-GCLC and anti-GCLM (Abcam, Cambridgeshire, UK) and mouse antibody anti-b-actin (Sigma) and anti-γ-H2AX (Abcam).
Anti-mouse and anti-rabbit secondary antibodies were coupled with horseradish peroxidase (GeHealthcare, Buckinghamshire, UK). Proteins were visualized with an enzyme-linked chemiluminescence detection kit according to the manufacturer's (GeHealthcare) instructions. Chemiluminescence was monitored by exposure to film and the signals were analyzed under non-saturating conditions with an image densitometer connected to Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
Anti-mouse and anti-rabbit secondary antibodies were coupled with horseradish peroxidase (GeHealthcare, Buckinghamshire, UK). Proteins were visualized with an enzyme-linked chemiluminescence detection kit according to the manufacturer's (GeHealthcare) instructions. Chemiluminescence was monitored by exposure to film and the signals were analyzed under non-saturating conditions with an image densitometer connected to Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
8
Red Cabbage Bioactivity Evaluation
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Red cabbage (Brassica oleracea L. var capitata f. rubra) was purchased from a local supermarket, while Pectinase and Cyanidol-3-Glucoside were obtained from Aladdin (Shanghai, China). Hemp stalk was provided as a present. In addition, cationic hydroxyethyl cellulose (cHEC), CCK-8 (Cell Counting Kit-8), fluorescein isothiocyanate (FITC), and 2′,7′-dichlorohydrofluorescein diacetate (DCFH-DA) were acquired from Usolf (Qingdao, China), Dojindo (Dojindo Laboratories, Japan), and Sigma Chemical Co. (Sigma, USA), respectively. Anti-γH2AX was obtained from Abcam (Abcam, UK). DMEM, penicillin, and streptomycin solution were purchased from HyClone (GE Health Care Life Science, USA), while B27 was obtained from Gibco (Thermo Fisher, USA). BALB/c mice and nude mice were purchased from Dashuo Biological Technology Co., Ltd. (Chendu, China).
9
Western Blot Analysis of Cellular Markers
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Cell lysates were prepared with Laemmli buffer supplemented with protease inhibitors (complete tablet; Roche) and phosphatase inhibitors (PhosSTOP tablet; Roche) and analysed by Western blotting with the following primary antibodies: anti-γH2AX (Abcam); anti-PTB (Abcam); anti-nPTB (Abcam); anti-βIII Tubulin (Abcam); anti-Tyrosine Hydroxylase (Abcam); anti-GAPDH (Thermo Scientific); anti-MAP2 (Novusbio); anti-DROSHA (Cell Signaling); anti-DICER (Sigma); anti-H2AX (Millipore). Primary antibodies were revealed with peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (Jackson Immunoresearch Laboratories) and enhanced chemiluminescence system (Super Signal West Pico Pierce or Super Signal West Dura Extended). βIII Tubulin antibody was revealed with alkaline phosphatase-conjugated goat anti-chicken antibody (Santa Cruz) with a colorimetric assay.
10
Immunofluorescence Characterization of L1 and IAP
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Rabbit polyclonal L1 ORF1 antisera were made through immunization of rabbits with recombinant ORF1 protein. The following antibodies were used at the indicated dilutions for IF: anti-G9a (1:50) (A. Tarakhovsky, The Rockefeller University, New York, NY, USA), anti-ORF1 L1 (1:500), mouse monoclonal anti-GLP (R&D systems, Minneapolis, MN, USA, PP-B0422-00) (1:100), anti-IAP Gag (1:500) (B. Cullen, Duke University, Durham, NC, USA), mouse monoclonal anti-γH2AX (Abcam, Cambridge, UK ab26350) (1:500), mouse monoclonal anti-H3K9me2 (Abcam, Cambridge, UK ab1220) (1:100) and rabbit polyclonal anti-Plzf (Santa Cruz, Dallas, TX, USA sc-22839) (1:100). Immunofluorescence and histology were performed as described [3 (link)]. Quantification of L1 Orf1 and IAP signal from at least 20 to 40 cells was performed using Fiji software (http://fiji.sc/Fiji ).
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