Ab7792

Manufactured by Abcam

Sourced in United Kingdom

Ab7792 is a laboratory antibody product designed for use in various research applications. The core function of this product is to detect and bind to a specific target molecule, providing researchers with a tool for investigating biological processes or detecting the presence of a particular analyte. No further details on the intended use or application of this product can be provided in an unbiased and factual manner.

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Lab products found in correlation

7 protocols using ab7792

Validating DUSP6 Knockdown by Western Blot

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To confirm the knockdown of DUSP6, total protein was harvested with RIPA buffer 48 h post siRNA transfection. Proteins (20 μg per sample) were separated by SDS-PAGE via an 8 % Precise protein gel (Life Technologies) and blotted onto a Immobilon-P transfer membrane (Millipore). Detection of DUSP6 was performed using mouse monoclonal antibody (1:1000; Abcam ab54940) and HRP-conjugated rabbit anti-mouse IgG (H+L) secondary antibody (1:5000; Jackson Immunoresearch 315-035-003), and finally visualized with SuperSignal West Dura Extended Duration Substrate (Life Technologies). The antibody was afterwards removed from the membrane with 10 M urea, 45 mM SDS followed by washing steps with 50 % ethanol, 10 % acetic acid. Consecutively, the membrane was re-probed with a mouse monoclonal anti-beta tubulin antibody (1:1000; Abcam ab7792) and the same secondary antibody as before to confirm equal loading amounts of protein lysate.

Wittig-Blaich S., Wittig R., Schmidt S., Lyer S., Bewerunge-Hudler M., Gronert-Sum S., Strobel-Freidekind O., Müller C., List M., Jaskot A., Christiansen H., Hafner M., Schadendorf D., Block I, & Mollenhauer J. (2017). Systematic screening of isogenic cancer cells identifies DUSP6 as context-specific synthetic lethal target in melanoma. Oncotarget, 8(14), 23760-23774.

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Cell Lysis and Protein Fractionation

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Cells were lysed in 50 mM tris, 40 mM NaCl, 2 mM MgCl₂, 0.5% Triton X-100, 1× protease inhibitors cocktail, and benzonase nuclease (25 U/ml) for 1 hour at 4°C. Cell lysate was cleared by centrifugation for 10 min at 4°C. Total protein concentration was determined using Bradford assay and fractionated by SDS–polyacrylamide gel electrophoresis. The following primary antibodies were used: 53BP1 (A300-273A, Bethyl Laboratories), β actin (AC-40, Sigma-Aldrich), β tubulin (ab7792, Abcam), GFP (ab290, Abcam), OXPHOS antibody cocktail (ab110411, Abcam), SOD1 (FL-154, Santa Cruz Biotechnology), TDP1 (ab4166, Abcam), TOP1 (C-21, Santa Cruz Biotechnology), VDAC1/Porin (ab15895, Abcam), and UQRC2 (ab14745, Abcam).

Chiang S.C., Meagher M., Kassouf N., Hafezparast M., McKinnon P.J., Haywood R, & El-Khamisy S.F. (2017). Mitochondrial protein-linked DNA breaks perturb mitochondrial gene transcription and trigger free radical–induced DNA damage. Science Advances, 3(4), e1602506.

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Enrichment and Analysis of Oxidized Proteins

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Azido-tagged biotin with or without a photocleavable linker (biotin-UV-azide and biotin-azide) were provided by Professor Ned A. Porter ^{6 (link)} (Supplementary Figure 10). The synthesis of these reagents is described in the Supplementary Material of Reference 6. DYn-2 was purchased from Cayman chemicals. DYn-2-d6 was synthesized as described in the Supplementary Methods. Strong cation exchange (SCX) spin columns were purchased from Nest group. Streptavidin sepharose was purchased from GE Healthcare Life Sciences. HPLC-grade water, acetonitrile (ACN), and methanol were purchased from J.T.Baker. Other chemicals and reagents were obtained from Sigma unless otherwise indicated. Antibodies against ACLY (ab40793, diluted at 1:5000), ACTN (ab6276, diluted at 1:5000), CFL1 (ab42824, diluted at 1:1000), EEF1G (ab72368, diluted at 1:5000), MAPK1/ERK2 (ab124362, diluted at 1:1000), RPL18 (ab166711, diluted at 1:1000), SIRT6 (ab88494, diluted at 1:2500), TUBB (ab7792, diluted at 1:5000), PRDX-SO_2/3H (ab16830, diluted at 1:2000), PRDX1 (ab15571, diluted at 1:2000), and PRDX2 (ab15572, diluted at 1:2000) were purchased from Abcam. Antibodies against HSP90 (CS#4874S, diluted at 1:1000) and YWAHE/14-3-3ε (CS#9635S, diluted at 1:1000) were purchased from Cell Signaling Technology.

Yang J., Gupta V., Carroll K.S, & Liebler D.C. (2014). Site-specific mapping and quantification of protein S-sulfenylation in cells. Nature communications, 5, 4776.

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Western Blot Analysis of Protein Targets

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Proteins were separated by SDS-PAGE and transferred to PVDF. Membranes were incubated with primary antibody solutions in blocking buffer. Primary antibodies used include: keratin 19 (mAb3238, Millipore, UK); ApoA1(Ab48647, Abcam, UK), M2PK (Ab38327, Abcam, UK); GAPDH (AM4300, Ambion); and α-tubulin (Ab7792, Abcam). Cross-reactions were visualised using HRP-conjugated secondary antibodies (Dako), Immobilon Western HRP substrate (Millipore, UK). A Chemigenius Bioimaging system was employed for band visualisation and densitometric analysis.

Evans C.A., Rosser R., Waby J.S., Noirel J., Lai D., Wright P.C., Williams E.A., Riley S.A., Bury J.P, & Corfe B.M. (2015). Reduced keratin expression in colorectal neoplasia and associated fields is reversible by diet and resection. BMJ Open Gastroenterology, 2(1), e000022.

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Enrichment and Analysis of Oxidized Proteins

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Yang J., Gupta V., Carroll K.S, & Liebler D.C. (2014). Site-specific mapping and quantification of protein S-sulfenylation in cells. Nature communications, 5, 4776.

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Cell Lysis and Protein Analysis

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Cell lysates were prepared by solubilising cell populations on ice for 20 minutes in lysis buffer; 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM DTT and 1 mM EDTA supplemented with 50 U/μl benzonase (Novagen), protease and phosphatase inhibitors (Sigma). Cleared lysates were produced by centrifugation of the resulting samples at 16,000 × g for 15 min at 4 °C. Gel electrophoresis was performed using the NuPAGE system (Invitrogen). Briefly, samples were resolved on 4–12% Bis-Tris gels in MOPS buffer, transferred to a PVDF membrane which was then probed for the protein of interest using antibodies diluted in TBS containing 5% Marvel and 0.1% Tween-20 (Sigma). Antibodies used were: alpha-Tubulin (Abcam ab7792, 1:1000), pATM (ab36810), β-actin (Abcam; ab8226), Cyclin B1 (Cell Signaling; mAb4135), EBLN1 (in-house synthesis and purification of a rabbit polyclonal antibody through Sheaf Innovations/BioServUK Ltd. using the peptide MSRPRNNPQTSSPQD), FLAG (Sigma; F3165), FLAG-HRP (Sigma; A8592), GFP (Abcam; ab290), anti-Histone H3 pSer10 (Cell Signaling; 9701), γH2AX (Cell Signalling; 25775), securin (Abcam; ab79546), TPR (ab58344, ab170940 and ab70610), HRP-secondary antibodies (DAKO, FITC) and Alexa-Fluor antibodies (Invitrogen).

Myers K.N., Barone G., Ganesh A., Staples C.J., Howard A.E., Beveridge R.D., Maslen S., Skehel J.M, & Collis S.J. (2016). The bornavirus-derived human protein EBLN1 promotes efficient cell cycle transit, microtubule organisation and genome stability. Scientific Reports, 6, 35548.

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Pancreatic Protein Extraction and Western Blot

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Pancreatic tissues were frozen at -80° C until homogenization in extraction buffer (100 mg/ml) on ice. The extraction buffer contained 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Igepal® CA-630, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 50 μM sodium orthovanadate (all from Sigma-Aldrich), and a protease inhibitors cocktail (Sigma-Aldrich) at a concentration of 4 μl/ml.
The following antibodies were used: anti-PGC-1± (1/500) (2178, Cell Signaling Technology, Danvers, MA, USA), anti-GCLc (1/1000) (ab41463, Abcam, Cambridge, UK), anti-NF-º B p65 (1/1000) (8242, Cell Signaling Technology), anti-phospho-NF-º B p65 (Ser 536) (1/1000) (3039, Cell Signaling Technology), anti-histone 3 (1/1000) (ab5103, Abcam), anti-phospho-STAT3 (Tyr705) (1/1000) (9145, Cell Signaling Technology), anti-STAT3
(1/1000) (4904, Cell Signaling Technology) and anti-beta tubulin (1/1000) (ab7792, Abcam).

Pérez S., Rius-Pérez S., Finamor I., Martí-Andrés P., Prieto I., García R., Monsalve M, & Sastre J. (2019). Obesity causes PGC-1α deficiency in the pancreas leading to marked IL-6 upregulation via NF-κB in acute pancreatitis. The Journal of pathology, 247(1).

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