Parotid glands from wild type and Prkcz−/− mice were lysed in RIPA buffer with 5 mM sodium orthovanadate (Fisher Scientific, Waltham, MA), protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and 100 mM PMSF (Pierce/Thermo Scientific, Rockford, IL); then, samples were sonicated for 2 minutes and boiled for 10 min until homogeneous. 100 ug of protein from supernatant were loaded in 12% polyacrylamide gels. Transference was performed for 1 hour at 100 V using 0.45 μm Immobilon-P membranes (Millipore, Billerica, MA). After blocking in TBST buffer containing 2% BSA, membranes were incubated at 4 °C overnight in primary antibody at 1:1000 dilution. For detection, HRP conjugated secondary antibodies were applied to the membranes for 1 hour at room temperature and ECL substrate (Pierce/Thermo Scientific) was used following instructions by the manufacturer. The following antibodies were used: anti-total aPKCζ (Cell signaling, #9368), anti-PKCζ (phospho T560) antibody [EP2037AY] (Abcam, ab62372), Anti-Beta-tubulin (Thermo Scientific, #rb-9249-p), anti-Yap (D8H1X) XP Rabbit mAb (Cell Signaling, # 14074), anti-Rabbit-HRP (1:2000, Cell signaling, #7074 S), and Goat-anti-rabbit (1:10000, BioRad, #972-4446).
Anti beta tubulin
Manufactured by Thermo Fisher Scientific
Sourced in Australia
Anti-beta tubulin is a laboratory reagent used to detect and quantify the presence of beta-tubulin in biological samples. It is a primary antibody that specifically binds to the beta-tubulin protein, which is a key component of the cytoskeleton in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (4 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Sodium orthovanadate, by Thermo Fisher Scientific (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Thermo Fisher Scientific (2 mentions)
Goat serum, by Thermo Fisher Scientific (2 mentions)
Anti hec1, by Thermo Fisher Scientific (2 mentions)
Phalloidin tritc, by Santa Cruz Biotechnology (2 mentions)
Goat anti mouse igg alexa fluor 405, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (2 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Restore western blotting stripping buffer, by Thermo Fisher Scientific (1 mentions)
Coomassie plus the better bradford assay, by Thermo Fisher Scientific (1 mentions)
Anti phospho c jun s63, by Cell Signaling Technology (1 mentions)
Donkey anti human igg alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Nhs biotin, by Merck Group (1 mentions)
3h thymidine, by PerkinElmer (1 mentions)
Anti phosphopaxillin, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti beta tubulin
1
Western Blot Analysis of aPKCζ in Parotid Glands
2
Western Blot Analysis of Parotid Gland Proteins
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Whole protein lysates from parotid glands of FVB, wild type C57BL/6J and Prkcz-/- mice were harvested and processed for immunoblotting as previous described [19 (link),36 (link)]. Primary cell lysates were processed in the same fashion. Briefly, samples were lysed in RIPA buffer with 5mM sodium orthovanadate (Fisher Scientific, Hampton, NH), protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and 100mM PMSF (Thermo Scientific, Waltham, MA). The Coomassie Plus-The Better Bradford Assay (Thermo) was used to determine protein concentrations and 30–100μg of total lysate was used. The following antibodies were used: anti-PARD3 (Abcam), anti-PARD6 (Proteintech), anti-total PKCζ (Cell Signaling), anti-pPKCζ (T560) (Abcam), anti-phospho-c-Jun (S63) (Cell Signaling), and anti-beta-tubulin (Thermo Scientific). Restore Western Blotting Stripping Buffer (Fisher) was used to strip membranes and reprobed for loading controls.
3
Visualizing Mitotic Spindle and Kinetochore Dynamics
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Cells synchronized with the Cdk1 inhibitor RO-3306 were released with a drug washout and monitored for mitotic entry. Cells were fixed with 4% paraformaldehyde (15 minutes) after ~ 1 hour after drug washout, where the maximal number of cells were observed (by visual inspection) to be in metaphase. Fixed cells were permeabilized in 0.1% Triton X-100 solution for 15 minutes and blocked with 5% goat serum (Invitrogen, Grand Island, NY) for 45 minutes. Primary antibodies were diluted in an antibody dilution buffer consisting of PBS with 1% Bovine Serum Albumin and Triton-X 100 and stored overnight at 4° C. Primary antibodies include Anti-beta tubulin (1:500, mouse monoclonal, 2 28 33, Invitrogen) and Anti-Hec1 (1:1000, human monoclonal) for labeling microtubules and kinetochores respectively. Secondary antibodies diluted in antibody dilution buffer were added along with the conjugated Phalloidin-TRITC (Santa Cruz Biotechnology) or Alexa Fluor 647 Phalloidin (1:40–1:80, Invitrogen) and stored in a dark place for 45 minutes. Secondary antibodies include donkey anti-human IgG Alexa Fluor 555 (1:600) and Goat anti-mouse IgG Alexa Fluor 405 (1:500, Invitrogen). Confocal microscopy was performed using a laser scanning confocal microscope (LSM 880, Carl Zeiss Inc.) with optimal imaging settings and z-slice thicknesses ranging from 0.36–0.5 μm.
4
Insulin and IGF-1 Receptor Signaling
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Insulin was purchased from Lyppard Australia Pty Ltd. IGF-II and (His4, Tyr15, Thr49, Ile51) IGF-I (qIGF-I) were produced in-house as described by Ref. (7 (link)) and the S597 peptide was provided by Dr. L. Schäffer, Novo Nordisk Denmark. Hybridoma cells expressing antibodies specific for the IR alpha subunit (83-7) and beta subunit (CT-1) were a kind gift from Siddle (34 (link), 35 (link)). Anti-phospho IGF-1R/IR Y1158, Y1161, Y1162 (p3Y = pY1146, pY1150, pY1151 IR-A numbering), anti-phospho IR Y960 (pY960), and anti-beta tubulin were from Invitrogen (Life Technologies, Mulgrave, VIC, Australia). Anti-phospho IR Y1316 and anti-phospho Y1322 and the Pathscan® Multiplex Western Cocktails were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Cy3 AffiniPure Donkey Anti-Mouse IgG and Cy5 AffiniPure Donkey Anti-Rabbit IgG were purchased from Jackson/Abacus ALS. Europium-labeled anti-phosphotyrosine antibody (Eu-pY20), europium-labeled-streptavidin (Eu-SA) and [3H] thymidine were purchased from PerkinElmer Life Sciences. hIR-A overexpressing R− fibroblast cells (derived from IGF-1R knockout mouse embryonic fibroblasts) were produced by (6 (link)). hIR-A overexpressing L6 myoblasts were kindly provided by Dr. B. F. Hansen (Novo Nordisk A/S, Denmark). Protease inhibitor cocktail and NHS-Biotin were from Sigma.
5
Immunofluorescence Labeling of Cytoskeleton
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Cells were fixed with 4% paraformaldehyde for 15 minutes. Following 2 times PBS wash, permeabilization was performed with a 0.1% Triton X-100 solution. Permeabilized cells were washed with PBS (2x) and blocked with 5% goat serum (Invitrogen, Grand Island, NY) for 45 minutes. Primary antibodies were diluted in an antibody dilution buffer consisting of PBS with 1% Bovine Serum Albumin and Triton-X 100, and stored overnight at 4 o C. Primary antibodies include Anti-beta tubulin (1:500, mouse monoclonal, 2 28 33, Invitrogen), Anti-Hec1 (1:1000, human monoclonal) and Anti-phospho-Paxillin (1:100, rabbit polyclonal, pTyr31, Invitrogen). Secondary antibodies diluted in antibody dilution buffer was added along with the conjugated Phalloidin-TRITC (Santa Cruz Biotechnology) or Alexa Fluor 647 Phalloidin (1:40-1:80, Invitrogen), and stored in a dark place for 45 minutes. Secondary antibodies include donkey anti-human IgG Alexa Fluor 555 (1:600), Goat anti-mouse IgG Alexa Fluor 405 (1:500, Invitrogen) and Goat anti-mouse IgG Alexa Fluor 647 secondary antibody (1:500, Invitrogen). Confocal microscopy was performed using a laser scanning confocal microscope (LSM 880, Carl Zeiss Inc.) with optimal imaging settings and z-slice thicknesses ranging from 0.36-0.5 µm.
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