Cells were lysed in RIPA buffer supplemented with 1 mM Na3VO4, 1 mM cOmplete ULTRA Tablets, Mini, EDTA-free EASYPack (Roche), and 100 nM MG-132. Protein concentrations were measured using a DC Assay Kit (Bio–Rad), and 100 μg of protein was used for each sample. Samples were added to either mouse IgG magnetic beads (Cell Signaling) or to 6 μg of primary antibody and incubated at 4 °C overnight with gentle agitation. Samples containing antibody were added to cleared protein G beads (with active motifs) and incubated for 4 h at 4 °C with gentle agitation. All samples were then washed with TBS, and proteins were eluted from the beads by adding 2× nonreducing Laemmli buffer (250 mM Tris-HCl; 8% SDS; and 40% glycerol, pH 6.8) and incubation at 95 °C for 5 min. β-Mercaptoethanol was added to each sample, and immunoblotting was performed.
Ultra tablets
Manufactured by Roche
Sourced in Switzerland
ULTRA tablets are laboratory equipment used for sample preparation and analysis. They provide a compact and efficient solution for tasks requiring precise temperature control and mixing. The core function of ULTRA tablets is to facilitate controlled temperature regulation and mixing of samples in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Easypack, by Roche (2 mentions)
β actin, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dc assay kit, by Bio-Rad (1 mentions)
Mouse igg magnetic beads, by Cell Signaling Technology (1 mentions)
Ab18058, by Abcam (1 mentions)
Tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Pc130, by Merck Group (1 mentions)
Mini protean tgx 4 15 precast gels, by Bio-Rad (1 mentions)
Sc 365341, by Santa Cruz Biotechnology (1 mentions)
Spm227, by Abcam (1 mentions)
Ha 11 clone 16b12, by Fortrea (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Pan akt, by Cell Signaling Technology (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Phosstop easypack, by Roche (1 mentions)
Ecl prime immuno blotting detection reagent, by GE Healthcare (1 mentions)
7 protocols using ultra tablets
1
Immunoprecipitation and Immunoblotting Protocol
2
Western Blot Analysis of DNA Repair Proteins
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For Western blot analysis, the cells were lysed in buffer containing 20 mM Tris–HCl (pH 7.5), 1 mM Na2EDTA, 1 mM EGTA, 150 mM NaCl, 1% (w/v) NP40, 1% sodium deoxycholate, 2.5 sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM NA3VO4 and 1 μg/ml leupeptin supplemented with cOmplete ULTRA Tablets (Roche, Basel, Switzerland). Denatured proteins (20–40 μg) were electrophoresed in 9% SDS-PAGE gels or MiniPROTEAN® TGX™ 4–15% Precast gels (Bio-Rad, Hercules, CA, USA) or on 3–8% Tris-acetate gels (Thermo Fisher Scientific, Waltham, MA, USA) transferred onto a nitrocellulose membrane and probed with specific antibodies, including anti-Vinculin (1/8000, SPM227, ab18058, Abcam, Cambridge, UK), anti-RAD51 (1/1000, Ab-1, PC130, Millipore, Burlington, MS, USA), anti-RAD52 (1/500, sc-365341, Santa Cruz, Dallas TX, USA), anti BRCA2 (1/500, OP95, Millipore, Burlington, MS, USA), anti-PALB2 (1/1000, GTX85263, Genetex, Irvine, CA, USA) and anti-HA (1/1000, HA.11 clone 16B12, Covance, Princeton, NJ, USA). Immunoreactivity was visualized using an enhanced chemiluminescence detection kit (ECL, Pierce).
3
Quantifying mTOR Signaling in Brain Regions
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The hippocampus, cortex and cerebellum from control and Raptor fb-KO mice were isolated, homogenized in cold homogenizing buffer [200 mM HEPES, 50 mM NaCl, 10% Glycerol, 1% Triton X-100, 1 mM EDTA, 50 mM NaF, 2 mM Na3VO4, 25 mM β-glycerophosphate, and EDTA-free complete ULTRA tablets (Roche, Indianapolis, IN)] and centrifuged at 13,000 × g for 10 min. The supernatants (of 30 µg of protein/sample) were resolved on SDS–PAGE (10%) and transferred onto nitrocellulose membranes (Pall, Port Washington, NY). Treatment with DHPG followed by biochemical analysis, was performed as previously described4 (link). Briefly, after treatments, slices were snap-frozen on dry ice, then suspended in lysis buffer and analyzed by western blotting, which was performed as we previously described12 (link),19 (link). Antibodies against p-S6 (1:1000, Ser240/244 #5364), p-Akt (1:1000, Ser473 #9271), total S6 (1:1000 #2217), total Akt (1:1000 #9272), raptor (1:1000 #2280), and Rictor (1:1000 #2114) were purchased from Cell Signaling and Technology Laboratories (Danvers, MA) and β-actin (1:5000 #1501) from Millipore (Temecula, CA).
4
Quantifying mTOR Signaling in Brain Regions
5
Protein Extraction and Western Blot Analysis
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Samples were homogenized in buffer containing 200 mM HEPES, 50 mM NaCl, 10% glycerol (vol/vol), 1% Triton X-100 (vol/vol), 1 mM EDTA, 50 mM NaF, 2 mM Na3VO4, 25 mM β-glycerophosphate and EDTA-free complete ULTRA tablets (Roche). A total of 30 μg of protein per sample was resolved on SDS-PAGE (10%), transferred onto nitrocellulose membranes and blotted as previously described15 (link). Western blots were quantified by densitometry using the ImageJ software.
6
Ventral Tegmental Area Microdissection and Western Blotting
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VTA samples were micro-dissected from 1 mm coronal sections obtained using an acrylic mouse brain matrix (Stoelting). Briefly, mice were killed by an overdose of isoflurane and their brains were quickly removed and placed in ice-cold PBS-immersed brain matrix for sectioning. The section containing most of the VTA (typically 8th from rostral to caudal) was then transferred for microdissection of the VTA using scalpels over an ice-cold petri-dish. Samples were collected in pre-chilled microcentrifuge tubes and lysed in homogenizing buffer [200 mM HEPES, 50 mM NaCl, 10% Glycerol, 1% Triton X-100, 1 mM EDTA, 50 mM NaF, 2 mM Na3VO4, 25 mM β-glycerophosphate, and EDTA-free complete ULTRA tablets (Roche, Indianapolis, IN)]. Western blotting was performed as previously described (Huang et al., 2013 (link)). Primary antibodies for Western blotting were rabbit anti-p-eIF2α (Ser51) (1:1000, Cell Signaling Technology Laboratories, Danver, MA), mouse anti-total eIF2α (1:1000, Cell Signaling Technology Laboratories, Danver, MA), and mouse anti-β-actin (1:10,000, EMD Millipore, Billerica, MA).
7
Insulin Signaling Pathway Analysis
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After overnight fasting, mice were injected with 1 U/kg of human fast working insulin (Aspart, Novo Nordisk). After 30 min VAT, liver and muscle were isolated and snap-frozen in liquid nitrogen. Samples were lysed in Triton lysis buffer (50 mM HEPES, 1% Triton X-100, 10 mM EDTA, 0.1% SDS, 50 mM NaCl, complete ULTRA Tablets, Mini, Easypack (Protease inhibitor cocktail tablets) (Roche) and PhosStop Easypack (Phosphatase inhibitor cocktail tablets) (Roche) in a tissue homogenizer. Protein contents were determined by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and equal amounts of total lysate were analyzed by 12% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to Immobilon-P and incubated with blocking buffer (Tris buffered saline/Tween-20) containing 2% low-fat milk for 1 h before incubating with an antibody against the insulin receptor, p-Akt (Ser 473 ) and pan-Akt from Cell Signaling. Bands were visualized with ECL Prime Immuno Blotting Detection Reagent (GE Healthcare) using ImageQuant LAS 4000mini (GE Healthcare, Life Science). Density of bands was calculated relative to pan-Akt using ImageJ software.
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