Total protein was isolated from cultured cells using RIPA lysis buffer (Beyotime, Beijing, China) and then quantitated by the BCA assay kit (Beyotime, Beijing, China). Twenty micrograms of total protein were mixed with 2× loading buffer, separated on SDS/10% PAGE, and then transferred to 0.2 μm PVDF membranes (Millipore, Bedford, MA, U.S.A.). The membranes were blocked in 5% non-fat milk dissolved in TBS and Tween 20 (TBST) buffer for 1 h and incubated with primary antibodies overnight at 4°C. Antibodies against GAPDH (sc-32233, mouse monoclonal) were purchased from Santa Cruz Biotechnology (CA, U.S.A.), and antibodies against MDM2 (ab38618, rabbit polyclonal), CDK6 (ab3126, mouse monoclonal), and GADD45A (ab180768, rabbit polyclonal) were purchased from Abcam (Cambridge, U.K.). Finally, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, CA, U.S.A.; sc-2004, goat anti-rabbit IgG-HRP; sc-2005, goat anti-mouse IgG-HRP), and the immunoactivity was detected using ECL-Plus kit (Amersham Biosciences, Piscataway, NJ, U.S.A.).
Ab38618
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab38618 is an antibody that can be used for laboratory research purposes. It is a protein that recognizes and binds to a specific target molecule.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (4 mentions)
Ab180768, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab9485, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ecl plus kit, by Cytiva (1 mentions)
0.2 μm pvdf membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Ab33911, by Abcam (1 mentions)
Ab154856, by Abcam (1 mentions)
Ab185656, by Abcam (1 mentions)
Ab133504, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Boster Bio (1 mentions)
Ab133534, by Abcam (1 mentions)
17 protocols using ab38618
1
Protein Isolation and Western Blotting Analysis
2
Western Blot Analysis of Cell Signaling Proteins
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After 0 μM, 0.5 μM, 1.0 μM, or 1.5 µM 7f treatment of A549 or PC-9 cells, performed western blot analysis according to relevant general operating procedures. Primary antibodies (100 μL/cm2) included those against c-myc (ab185656, Abcam Company, USA), cyclin D (ab16663, Abcam Company, USA), cyclin E (ab33911, Abcam Company, USA), Ki67 (ab16667, Abcam Company), P53 (SC-126, Santa Cruz Company, USA), MDM2 (ab38618, Abcam Company, USA), cleaved caspase-3 (YT6161, Immunoway Company, USA), caspase-3 (YT6113, Immunoway Company, USA), bax (50599–2-lg, Proteintech Company, USA), VEGFR-2 (#9698, Cell Signaling Technology, USA), β-actin (60012–1-lg, Proteintech Company, USA), bcl-2 (12789–1-AP, Proteintech Company, USA), VDCA-1 (ab154856, Abcam Company, USA), p-VEGFR-2 (#3770, Cell Signaling Technology, USA), and Cytochrome C (ab133504, Abcam Company, USA).
3
Subtype-specific Biomarkers in Esophageal Adenocarcinoma
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To identify subtype-specific biomarkers, tissues from 237 primary EAC cases were collected from 2011–2017 at the First Affiliated Hospital of Zhengzhou University and subjected to tissue microarray (TMA). Ethics approval was granted by the hospital’s institutional review board. Antibodies against MSH2 (1:200; D24b5; CST, United States), MSH6 (1:500; 3E1; CST, United States), and MDM2 (1:500; ab38618; Abcam, China) were used in the immunohistochemical analysis, as these corresponded to the three top-ranked subtype-specific genes for which high-quality commercial antibodies were available based on the SAM-seq results. The immunohistochemistry (IHC) was performed according to Wang et al. (2019) (link). In brief, the TMA sections were baked in an oven for 1 h, deparaffinized, and rehydrated with xylene and graded alcohols. After blocking by 3% H2O2 for 15-min incubation, the sections were boiled in antigen retrieval buffer. Then, the TMAs were incubated in primary antibodies and HRP-conjugated secondary antibodies (Boster). After developing with diaminobenzidine, the sections were counterstained with hematoxylin and dehydrated in graded alcohols and xylene. Abcam, CST) were used. The immunohistochemical staining was graded as follows: 0, no staining; 1, weak staining (10% of cells); 2, moderate staining (10–30%); and 3, strong staining (> 30%).
4
Western Blot Analysis of Cell Signaling Proteins
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Total proteins from frozen tissues or cells were extracted using RIPA buffer containing a 1% protease inhibitors (Roche). Protein concentration was determined by Bradford assay (Thermo Scientific, Waltham, MA, USA). Equal amount of proteins were separated in 12% SDS-polyacrylamide gels and transferred onto PVDF membranes. Next, we blocked the membrane with 5% non-fat dry milk in Tris-buffered saline and Tween 20 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl and 0.05% Tween, TBS-T), and incubated the membrane at 4 °C overnight with primary antibodies, followed by with horseradish peroxidase (HRP)-conjugated secondary antibodies. The immunoreactivities were visualized by enhanced chemiluminescence (ECL) kit according to manufacturer’s instruction. Western blot analyses were performed using primary antibodies against TNFAIP8 (ab166804, Abcam, Cambridge, MA, USA), MDM2 (ab38618, Abcam, Cambridge, MA, USA), p53 (ab179477, Abcam, Cambridge, MA, USA), cyclin D1 (ab134175, Abcam, Cambridge, MA, USA), CDK6 (ab124821, Abcam, Cambridge, MA, USA) and RAD51 (ab133534, Abcam, Cambridge, MA, USA). Western blot bands were quantified by the ImageJ software (U.S. National Institutes of Health, USA). The experiment was repeated thrice.
5
Protein Expression Analysis of miR-1305
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Cells were harvested after the transfection of miR-1305 and control miRNA for 48 hrs. Cells were lysed with the RIPA buffer (Beyotime, Shanghai, China) and the protein concentration was quantified using the BCA protein assay kit (Beyotime, Shanghai, China). Equal amount of protein was separated by SDS-PAGE and transferred onto the PVDF membrane (GE Healthcare, USA). After blocking with 5% non-fat milk, the membrane was incubated with the primary antibody overnight at 4ºC followed by the secondary HRP-conjugated antibody for 1 hr at RT. These antibodies used in this study including anit-MDM2 (ab38618; Abcam, USA), anti-p53 (ab32389; Abcam, USA) and anti-GADPH (ab9485; Abcam, USA) were commercially purchased. The protein bands were analyzed using the ImageQuant LAS 4000 min Imaging System (GE Healthcare, USA).
6
Western Blot Analysis of Phospho-Akt and Phospho-MDM2
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The cell monolayers were washed with PBS and lysed at the indicated times. The lysates were collected and incubated on ice for 10 min. The lysates were cleared by centrifugation at 10,000g for 5 min at 4 °C. The supernatants were analyzed for total protein content with a BCA protein-assay kit (Beyotime). The total protein (30 μg) was resolved by 10% SDS–PAGE and transferred onto nitrocellulose membranes (Millipore). The membranes were blocked with 5% skim milk for 1 h at 37 °C and then incubated overnight at 4 °C with specific antisera: rabbit anti-phospho-Akt antibody (Ser473) (ab81283, Abcam), rabbit anti-Akt antibody (ab32505, Abcam), rabbit anti-phospho-MDM2 antibody (Ser166) (ab170880, Abcam), rabbit anti-MDM2 antibody (ab38618, Abcam), rabbit PI3K p85 antibody (ab40755, Abcam), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GADPH) antibody (ab22555, Abcam) and rabbit anti-p53 antibody (9282, CST). After three rinses in TBST buffer, the membranes were incubated at 37 °C for 1 h with IRDye 800DX-conjugated anti-rabbit IgG (1:8000; Rockland Immunochemicals) diluted in TBST as a secondary antibody. The membranes were washed three times in PBST, then visualized and analyzed with an Odyssey infrared imaging system (LI-COR Biosciences).
7
Western Blot Analysis of Protein Expression
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Total protein was isolated using the Radioimmunoprecipitation Assay Kit (R0010, Beijing Solabio Life Sciences, Beijing, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis kit was used to prepare 10% separation gel and 5% concentrating gel samples. After electrophoresis, the protein was transferred onto a nitrocellulose membrane. The membrane was incubated with the following primary antibodies overnight at 4°C: rabbit polyclonal antibody to TPX2 (ab71816, 1:100, Abcam), p53 (ab32049, 1:1,000, Abcam), p21 (ab47300, 1:500, Abcam), MDM2 (ab38618, 1:1,000, Abcam), bax (ab53154, 1:500, Abcam), bcl-2 (ab59348, 1:1,000, Abcam), and PCNA (ab152112, 1:500, Abcam). The membrane was rinsed three times using Tris-buffered saline containing Tween 20 on the following day. The secondary rabbit polyclonal antibody (ab7312, Abcam) was added and incubated for 1 h. Developer (D-90G, Shanghai Yingdian Detection Equipment, Shanghai, China) was added, and images were acquired using the Bio-Rad gel imaging system (Beijing Thmoregan Biological Technology, Beijing, China). IPP7.0 software (Media Cybernetics, Bethesda, MD, USA) was adopted for quantitative analysis.
8
Isolation and Characterization of Gallbladder Cancer-associated Fibroblasts
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We purchased GBC‐SD, NOZ and SGC‐996 cell lines from Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured GBC cell lines with RPMI‐1640. All cells used in this study were authenticated by short tandem repeat DNA profiling by Genetica, and mycoplasma contamination was routinely tested using detection kit (CA1080, Solarbio).
We isolated CAFs and normal fibroblasts (NFs) from 4 different GBC patients (all these 4 patients with T3N0M0 adenocarcinoma) and 4 different chronic cholecystitis patients (all these 4 patients were young men less than 35 years old and with a history of gallstone less than one year), using previously described digestion methods.5 These antibodies and reagents were used in this study: anti‐NRP‐1 (Rabbit polyclonal, ab25998, Abcam), anti‐Murine double minute 2 (anti‐MDM2; Rabbit polyclonal, ab38618, Abcam), anti‐Cyclin A2 (anti‐CCNA2; Mouse monoclonal, BF683, Cell Signaling), anti‐Early Growth Response 1 (anti‐EGR1; Rabbit monoclonal, 44D5, Cell Signaling), anti‐IGF1R (Rabbit polyclonal, ab39675, Abcam), anti‐GAPDH (Mouse Monoclonal, AM4300, Invitrogen), anti‐IL‐8 (Mouse monoclonal, M801, Thermo), secondary antibody to Mouse IgG‐H&L (Goat polyclonal, ab150113, Abcam), IL‐8 receptors inhibitor Reparixin (HY‐15251, MCE) and Human IL‐8 ELISA Kit (KE00006, Proteintech Group).
We isolated CAFs and normal fibroblasts (NFs) from 4 different GBC patients (all these 4 patients with T3N0M0 adenocarcinoma) and 4 different chronic cholecystitis patients (all these 4 patients were young men less than 35 years old and with a history of gallstone less than one year), using previously described digestion methods.
9
Immunohistochemical Analysis of DLBCL
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For IHC, sections of DLBCL were incubated with primary anti-rabbit MDM2 (1:50; ab38618; Abcam, Cambridge, UK) and anti-rabbit CDC20 (1:200; Q105; Bioworld Technology Inc., St Louis Park, MN, USA) at 4 °C overnight. On the next day, the tissues were incubated with goat anti-rabbit secondary antibody (Zhongshan Goldbridge Biotechnology Co., Ltd., Beijing, China) at 37 °C for 1 h. All images were observed under a fluorescence microscope (IX73; Olympus, Tokyo, Japan).
10
Immunofluorescence Staining of Lymphoma Cells
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Herein, 4% paraformaldehyde-fixed cell suspensions of OCI-Ly3 and OCI-Ly10 were centrifuged in a Cytospin onto coverslips. The cells were permeabilized in 0.1% Triton X-100 for 5 min and blocked in 5% bovine serum albumin (BSA) at room temperature for 1 h, and then incubated for 1 h with primary anti-rabbit MDM2 (1:200, ab38618; Abcam, Cambridge, UK), anti-mouse p53 (1:200, DO-1, Santa Cruz) and anti-rabbit CDC20 (1:500; ab26483; Abcam, Cambridge, UK), washed thrice with PBS, and incubated with Alexa Fluor 488/555-conjugated secondary antibodies (Beyotime Institute of Biotechnology, Shanghai, China) at room temperature for 1 h in the dark. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime Institute of Biotechnology, Shanghai, China) for 5 min. Images were captured using a confocal microscope (LSM880, Carl Zeiss, Oberkochen, Germany).
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