Ab125243
Manufactured by Abcam
Ab125243 is a monoclonal antibody that can be used for the detection of a specific target protein in various applications such as Western blotting, immunohistochemistry, and ELISA. The product details and technical specifications are available on the Abcam website.
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12 protocols using ab125243
1
Co-immunoprecipitation of Protein Complexes
2
Multiparametric Immune Cell Profiling
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ELISA: The cell supernatant of feeder cells or cytotoxicity assay was collected to measure the levels of IFN-γ or Neo-2/15(Flag-tag) using their respective kits according to standard practice (R&D #DIF50C for IFN-γ, Abcam#ab125243 for Neo-2/15).
Flow cytometry: Flow cytometry was performed using the BD FACSAria system. Cells were incubated with indicated antibodies for 30 min at 4°C avoiding light. Cells were washed with DPBS and resuspended in FACS buffer before being subjected to FACS assay. Data were analyzed using FlowJo (version 10). The antibodies were from BioLegend and listed as follows: anti-CD107a-APC (328620), anti-OX40L-PE (326308), anti-CD56-PE (355504), anti-CD56-AlexaFluor488 (362518), anti-CD16-FITC (302006), anti-CD94-PE/Cy7 (305516), anti-NKG2A-APC (375108), anti-NKG2C-PE (375004), anti-NKG2D-FITC (320820), anti-KIR2DL2/L3-Percp/Cy5.5(312614), anti-KIR2DL1/S1/S3/S5-FITC (339504), anti-KIR3DL-BrilliantViolet421(312714), anti-NKp30-BrilliantViolet785 (325230), anti-NKp44-PE/Cy7 (325116), anti-NKp46-BrilliantViolet605 (137619).
Flow cytometry: Flow cytometry was performed using the BD FACSAria system. Cells were incubated with indicated antibodies for 30 min at 4°C avoiding light. Cells were washed with DPBS and resuspended in FACS buffer before being subjected to FACS assay. Data were analyzed using FlowJo (version 10). The antibodies were from BioLegend and listed as follows: anti-CD107a-APC (328620), anti-OX40L-PE (326308), anti-CD56-PE (355504), anti-CD56-AlexaFluor488 (362518), anti-CD16-FITC (302006), anti-CD94-PE/Cy7 (305516), anti-NKG2A-APC (375108), anti-NKG2C-PE (375004), anti-NKG2D-FITC (320820), anti-KIR2DL2/L3-Percp/Cy5.5(312614), anti-KIR2DL1/S1/S3/S5-FITC (339504), anti-KIR3DL-BrilliantViolet421(312714), anti-NKp30-BrilliantViolet785 (325230), anti-NKp44-PE/Cy7 (325116), anti-NKp46-BrilliantViolet605 (137619).
3
Western Blot Analysis of SOX6-FLAG
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Total extracts (15–30 μg/lane), resuspended in Laemmli buffer, were resolved by SDS/PAGE in a 10% acrylamide gel and blotted onto Hybond-ECL Nitrocellulose membrane (GE healthcare) under constant voltage at 100 V for 90 min at 4 °C (Biorad Transblot apparatus). Membranes were blocked for 1 hours at room temperature with Milk 5% in 1X TBS-T (Tris Buffered Saline, pH 7.6 and 0,1% Tween20) and incubated with the appropriate primary antibody diluted in Milk 5% 1X TBS-T overnight at 4°C. Membranes were washed three times in 1X TBS-T and incubated with the appropriate HRP-conjugated secondary antibody (in Milk 5% 1X TBS-T) for 1 hours at room temperature. After three washes in 1X TBS-T, antibodies binding was detected by ECL (Millipore). Antibodies used: anti-Flag F7425 (SIGMA) or ab125243 (abcam) and HRP-conjugated horse anti-mouse IgG #7076 (Cell Signaling), to detect the SOX6-FLAG protein; HRP-directly conjugated anti-beta actin #5125 (Cell Signaling) or anti-U2AF #U4758 (Sigma), to detect the loading control protein.
4
Co-immunoprecipitation of Protein Complexes
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Co‐immunoprecipitation was undertaken as documented,46 , 47 , 48 , 49 , 52 , 53 using antibodies against CNBP (sc‐515387, Santa Cruz Biotechnology, Inc.), KPNB1 (ab2811), SMARCC2 (ab243634), Flag‐tag (ab125243, Abcam Inc.), Myc‐tag (#2276), His‐tag (#9991) or GST‐tag (#2624, Cell Signaling Technology). After being released from the bead‐bound complex, proteins were determined via western blotting or mass spectrometric assays (Wuhan SpecAlly Life Technology Co., Ltd., China).46 , 47 , 52 , 53
5
Western Blot Analysis of Protein Expression
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For protein extraction, ground tissue or cells were lysed using RIPA lysis buffer, and the debris was removed by centrifuge. The protein in the supernatant was collected and quantified using a bicinchoninic acid assay (BCA) kit (Beyotime, China). The extracted proteins were separated on 12% SDS-PAGE gels, the voltage was first 80 V for ~30 min and subsequent 120 V, for ~60 min, and then and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA) (400 mA, 90 min). The membranes were blocked using skimmed milk in Tris-buffered saline Tween 20 (TBST) and probed using primary antibodies for 12 h at 4°C. After washing; the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for another 1 h at room temperature. Immunoreactive bands were detected using chemiluminescence detection kits (Millipore).
The primary antibodies used were as follows: SKP2 (ab183039, 1:500; Abcam, UK), β-actin (ab6276, 1:3,000; Abcam), LC3B (ab192890, 1:2,000; Abcam), p62 (#5114, 1:1,000; CST), PHLPP1 (ab135957, 1:1,000; Abcam), β-tubulin (#2146, 1:1,000; CST), AKT (#4691, 1:1,000; CST), phosphorylated-AKT (#4060, 1:2,000; CST), mTOR (#2972, 1:1,000; CST), phosphorylated-mTOR (#5536, 1:1,000; CST). Antibodies against HA tag (ab18181, 1:2,000; Abcam), Myc tag (ab9106, 1:1,000; Abcam), Flag tag (ab125243, 1:2,000; Abcam).
The primary antibodies used were as follows: SKP2 (ab183039, 1:500; Abcam, UK), β-actin (ab6276, 1:3,000; Abcam), LC3B (ab192890, 1:2,000; Abcam), p62 (#5114, 1:1,000; CST), PHLPP1 (ab135957, 1:1,000; Abcam), β-tubulin (#2146, 1:1,000; CST), AKT (#4691, 1:1,000; CST), phosphorylated-AKT (#4060, 1:2,000; CST), mTOR (#2972, 1:1,000; CST), phosphorylated-mTOR (#5536, 1:1,000; CST). Antibodies against HA tag (ab18181, 1:2,000; Abcam), Myc tag (ab9106, 1:1,000; Abcam), Flag tag (ab125243, 1:2,000; Abcam).
6
Co-immunoprecipitation of chromatin-modifying complexes
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Co-IP was carried out as reported previously56 (link), using 10 µg of antibodies for ARMC12 (ab72704, Abcam), RBBP4 (ab1765, Abcam), EZH2 (ab191250, Abcam), SUZ12 (ab12073, Abcam), FLAG (ab125243, Abcam), and HA (ab9110, Abcam). After releasing from bead-bound complex, protein levels were measured by western blotting. Uncropped images of blots were shown in Supplementary Fig. 10 . For proteomic analysis, proteins were digested by trypsin, and peptides were extracted according to Invitrogen protocol. LC-MS/MS detection was undertaken on a hybrid quadrupole-TOF LC/MS/MS mass spectrometer (TripleTOF 5600+, SCIEX, Redwood City, CA). For each scan cycle, one full-scan mass spectrum (with m/z ranging from 350 to 1500 and charge states from 2 to 5) and subsequent 40 MS/MS events were carried out. Analysis of MS/MS-acquired data was performed using ProteinPilot Software 5.0 and amino acid sequences in UniProt human protein database.
7
Western Blot Analysis of Pancreatic Cancer
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Western blot analysis was performed using standard techniques. Briefly, pancreatic cancer cells were digested and lysed by RIPA buffer supplemented with sodium orthovanadate, phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitors (Thermo Scientific). Protein concentration was determined by a BCA assay (Pierce). All lysates were separated by SDS‐PAGE, and then, the separated proteins were transferred to nitrocellulose membrane (Thermo Scientific). The membranes were blocked with 5% bovine serum albumin in PBS with 0.1% Tween 20 at 37°C for 2 h. The transferred membrane was blotted with the following primary antibodies: WT1 (1:5000, ab89901; Abcam); E‐cadherin (1:2000, ab40772; Abcam); HSP90 (1:2000, ab13492; Abcam); USP5 (1:2000, ab155993; Abcam); Flag (1:1000, ab125243; Abcam); HA (1:1000, AE008; Abclone Biotechnology); β‐actin (1:5000, ab8227, Abcam). All secondary antibodies are conjugated with horseradish peroxidase (HRP). Signals were detected by chemiluminescence reagents (Thermo Scientific).
8
Western Blot Analysis of Tumor Proteins
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Protein of tumor cells or tissues was prepared using 1× cell lysis buffer (Promega). Western blotting was performed as described previously52 (link)–55 (link), with antibodies specific for ARMC12 (ab72704, Abcam, Cambridge, MA, 1:1000 dilution), RBBP4 (ab1765, Abcam, 1:1000 dilution), EZH2 (ab191250, Abcam, 1:1000 dilution), SUZ12 (ab12073, Abcam, 1:500 dilution), H3K27me3 (ab6002, Abcam, 1:1000 dilution), HA (ab9110, Abcam, 1:1000 dilution), FLAG (ab125243, Abcam, 1:1000 dilution), CADM1 (ab3910, Abcam, 1:500 dilution), EGLN3 (ab30782, Abcam, 1:1000 dilution), HRK (ab45419, Abcam, 1:1000 dilution), HS6ST3 (SAB2101082, Sigma, 1:500 dilution), SMAD9 (ab115900, Abcam, 1:500 dilution), histone H3 (ab1791, Abcam, 1:1000 dilution), and GAPDH (ab8245, Abcam, 1:1000 dilution). Uncropped images of blots were provided in Supplementary Fig. 10 .
9
Estradiol-Inducible CgNLP1 Expression in Arabidopsis
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The vector pER8-CgNLP1-FLAG was constructed with an estradiol-inducible promoter and then transformed into Agrobacterium tumefaciens GV3101. The Agrobacterium-mediated flower dip method was used for Arabidopsis transformation of CgNLP1. T1 transgenic lines were screened with 30 mg/L hygromycin, and Western blot was used to confirm the positive transgenic lines after estradiol treatment. Appropriate total proteins from different lines were resolved on 10% polyacrylamide gels and transferred to a nitrocellulose membrane (Bio-Rad). Anti-FLAG monoclonal antibody (1:1,000, ab125243; Abcam) was used as a primary antibody to detect the expression of CgNLP1 in transgenic lines. Horseradish peroxidase-conjugated anti-mouse antibody was used as the secondary antibody, and the signal was detected using the ECL western detection kit (RPN2232; GE Healthcare).
10
Investigating RasGRP Signaling in Inflammatory Responses
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Wild-type C57BL/6 mice (6–8 weeks of age) were obtained from Jackson Laboratories (Bar Harbor, Maine). All the animal experiments were approved by the Medical Ethics Committee of the Zhejiang University School of Medicine and conducted according to the Declaration of Helsinki Principles. Poly (I:C) and LPS (0111:B4) were purchased from Sigma (St. Louis, MO). C-class CpG ODN (ODN 2395) was obtained from Invivogen (San Diego, California). Antibodies specific to Flag-tag (ab125243), phospho-RasGRP3 (Thr133) (ab124843), RasGRP1 (ab37927) and RasGRP2 (ab137608) were from Abcam Inc. (Cambridge, MA). Ab for RasGRP4 (sc-292930) was obtained from Santa Cruz Biotechnology (Dallas, TX). Ab specific for RasGRP3 (3334) and Abs against total and phosphorylated forms of ERK1/2 (Thr202/Tyr204) (4695, 9101), JNK1/2 (Thr183/Tyr185) (4668, 9252), p38 (Thr180/Tyr182) (9212, 9215), IKKα/β (Ser176/180) (2697, 8943), Akt (Ser473) (4060, 9272), Akt (Thr307) (9275) and IκBα (Ser32/36) (4814, 9246) were from Cell Signaling Technology (Beverly, MA). Ab for β-actin (A1987) was from Sigma.
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