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Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
Because of the use of antibodies in the final step, the name ‘immunoblotting’ is often used interchangeably, and is synonymous with Western blotting. The name ‘Western’ blotting coined by W Neal Burnette (41) is a pun on ‘Southern’ blot, which is a similar method for DNA developed by Edwin Southern in 1973 that was later published in 1975 (42).
Vesiculobullous Diseases
Published in Ayşe Serap Karadağ, Lawrence Charles Parish, Jordan V. Wang, Roxburgh's Common Skin Diseases, 2022
Snejina Vassileva, Kossara Drenovska
Laboratory studies: Similar to BP, the diagnosis is confirmed through histology and DIF microscopy. IIF can be negative in approximately 70% of cases. Antigen specificity of circulating antibodies may be defined using immunoblotting and immunoprecipitation and ELISA.
Measuring Oxidative Damage and Redox Signalling
Published in James N. Cobley, Gareth W. Davison, Oxidative Eustress in Exercise Physiology, 2022
James N. Cobley, Gareth W. Davison
Using omics workflows to measure exercise-induced oxidative damage is rate-limited by the time, expertise, and resources required. To expand current non-omic approaches to measure oxidative damage, we propose targeted immunological analysis. For example, manganese superoxide dismutase (MnSOD) activity is inhibited by tyrosine 34 (Y34) nitration to 3-nitrotyrosine (3-NT). Y34 nitration impairs catalysis by disrupting the hydrogen bond network required to protonate manganese bound superoxide (Abreu and Cabelli, 2010). MnSOD immunocapture followed by 3-NT blotting can assess a functionally annotated oxidative damage biomarker. However, appropriate steps to safeguard faithful immunocapture are required (e.g., covalently immobilising the antibody to a solid support). Additionally, reciprocal immunocapture is useful (e.g., confirming a 3-NT antibody elutes MnSOD). Further, immunoblotting is semi-quantitative. In certain cases, fluorophores could be used to achieve quantitative in-gel analysis. For example, a heterobifunctional hydrazine carbonyl reactive warhead with a short polyethylene glycol (PEG) spacer between a fluorescent moiety for quantitative in-gel fluorescent target protein carbonylation analysis.
The PERK/ATF4/CHOP signaling branch of the unfolded protein response mediates cisplatin-induced ototoxicity in hair cells
Published in Drug and Chemical Toxicology, 2023
Yanji Qu, Shimin Zong, Zhe Wang, Peiyu Du, Yingying Wen, Hao Li, Nan Wu, Hongjun Xiao
Total cellular protein was extracted by RIPA cell lysate buffer (Beyotime, #P0013D, China) containing 1% phenylmethanesulfonyl fluoride (PMSF, ST506, Beyotime, China). Protein concentration was determined using a BCA protein assay kit (P0012S, Beyotime, China). The western blot procedure was conducted as described in our previous study (Liu et al.2019a). Blots were incubated overnight at 4 °C with the primary antibodies. Following primary incubation, the membranes were incubated with HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (1:5000, SA00001-2, Proteintech, China) or HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (1:5000, SA00001-1, Proteintech, China) antibody for 1 h at room temperature. Immunoblotting was detected using the UVP ChemiDoc-It Imaging System (UVP, USA) and enhanced chemiluminescence detection kit (KF003, Affinity Biosciences, USA). The expression levels of proteins were quantified using ImageJ software (v1.51k, National Institutes of Health, USA) with β‑actin as the loading controls.
IRE1α overexpression in malignant cells limits tumor progression by inducing an anti-cancer immune response
Published in OncoImmunology, 2022
Adriana Martinez-Turtos, Rachel Paul, Manuel Grima-Reyes, Hussein Issaoui, Adrien Krug, Rana Mhaidly, Jozef P. Bossowski, Johanna Chiche, Sandrine Marchetti, Els Verhoeyen, Eric Chevet, Jean-Ehrland Ricci
For whole tumors, the frozen tissue was homogenized using a stainless-steel tissue grinder (1292, BioSpec Products). Tumor powder was lysed in a protease inhibitor-containing Laemmli or RIPA buffer using a Pre-cellys 24 tissue homogenizer (Bertin Instruments) (3 x 30s, 6500 x g). Magnetically isolated tumor cells (Tumor Cell Isolation kit for mice (Miltenyi Biotec, 130–110-187) were lysed in a protease inhibitor-containing RIPA buffer. Protein lyses were quantified and standardized (Pierce BCA protein assay kit, Thermo Scientific, #23225), and immunoblots were developed using the Amersham ECL Prime Western Blotting Detection Reagent (Cytiva, #RPN2236) and visualized with ImageQuant LAS 4000 (GE Healthcare, Life Science). Densitometry analyses were performed with the Multi Gauge v3 software. For in vitro cultured cells, Laemmli buffer for lysis and the XBP1s antibody (Biolegend #658802) were used while the XBP1 antibody (Santa Cruz #8015) was used for whole tumors lysed in Laemmli buffer and isolated tumor cells lysed in RIPA buffer. The following antibodies were used for immunoblotting:
Reciprocal SH2-SH3 Domain Contacts between ITK Molecules Limit T Cell Receptor Signaling in Th2-type CD4+ T Cells
Published in Immunological Investigations, 2022
Ji-Long Chen, Jennifer Y. Barr, Jonathan J. Zuk, Jacob V. Gorman, John D. Colgan
Transfected cells were harvested 30 hours after DNA was added and lysed by resuspending in NP-40 lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% NP-40) containing protease inhibitors (Roche Life Sciences, Indianapolis, IN, USA). For immunoprecipitations, 1 mg of protein was incubated with 8 µg of anti-Myc epitope tag antibody (clone 9E10; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hours at 4°C. Pre-washed Protein G Dynabeads (Invitrogen) were added and samples incubated for 1 hour at 4°C. After washing, beads were resuspended in NuPage sample buffer with reducing agent (Invitrogen) and heated to 70°C for 10 minutes followed by separation by SDS-PAGE. Transfer of proteins to PVDF membranes and immunoblotting were performed using standard techniques. Antibodies used for immunoblotting were: anti-Myc epitope tag antibody (clone 9E10; Santa Cruz), anti-FLAG epitope tag antibody (clone M2; Sigma-Aldrich), anti-Xpress epitope tag antibody (Invitrogen), anti-phosphotyrosine (clone 4G10; Millipore, Burlington, MA, USA), and anti-β-actin (Sigma-Aldrich, product #A2066).