Protein samples were solubilized with a loading buffer (4×) (2-mercaptoethanol + NuPage; 1:5) and heated at 95 °C for 5 min for reducing conditions, and for non-reducing conditions, the loading buffer was used without 2-mercaptoethanol. Then, proteins were separated by SDS-PAGE using 10% acrylamide gels and transferred to a nitrocellulose membrane (Bio-Rad Laboratories Inc., Hercules, CA, USA). Membranes were incubated overnight at 4 °C with the primary antibodies anti-CD63 (mouse,1:1000, Clone Ts63, Invitrogen, Waltham, MA, USA), anti-ALIX (mouse,1:500, Clone 3A9, Invitrogen), anti-TSG101 (mouse,1:500, Clone 4A10, Invitrogen), anti-α-tubulin (mouse, 1:10,000, Clone DM1A, Invitrogen), and anti-HSP90-B1 (rabbit,1:4000, CUSABIO), followed by one hour of incubation at room temperature with ECLTM Anti-mouse IgG and ECLTM Anti-rabbit IgG, Horseradish Peroxidase linker (GE Healthcare, Chalfont St Giles, UK). The membranes were visualized with ECLTM Prime Western blotting detection reagent (AmershamTM) in the ChemiDoc Bio-Rad instrument (Hercules, CA, USA).
Ecltm anti mouse igg
Manufactured by GE Healthcare
Sourced in United Kingdom
The ECLTM anti-mouse IgG is a laboratory equipment product. It is used to detect and quantify the presence of mouse immunoglobulin G (IgG) in samples.
Automatically generated - may contain errors
Lab products found in correlation
Ecltm anti rabbit igg, by GE Healthcare (5 mentions)
Anti hsp90 b1, by Cusabio (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Polyvinylidine fluoride membrane, by Merck Group (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Quick start bradford 1x dye reagent, by Bio-Rad (1 mentions)
Anti mre11 antibody, by Novus Biologicals (1 mentions)
Na934v, by GE Healthcare (1 mentions)
Nb100 142, by Novus Biologicals (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti erbb2, by Santa Cruz Biotechnology (1 mentions)
Anti foxo3a, by Cell Signaling Technology (1 mentions)
8 protocols using ecltm anti mouse igg
1
Western Blot Analysis of Extracellular Vesicle Markers
2
Protein Extraction and Western Blot Analysis
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Protein extraction from dissected tissues was performed as previously described [70 (link)]. Extracts were separated by electrophoresis in a 10% SDS-polyacrylamide gel at constant current of 120 volt. Proteins were transferred to polyvinylidinefluoride membranes (Millipore Corporation, Billerica, MA, USA), subsequently blocked 5% dry milk in TBST (Tris Buffered Saline with 0.1% Tween 20) for 1 h at room temperature and then incubated with anti-Dfr S/L, anti-Phm, anti-Dib, or anti-Actin (mAbcam 8224) as primary antibodies, and with ECLTM anti-rat IgG (Amersham), ECLTM anti-mouse IgG (GE Healthcare), and ECLTM anti-rabbit IgG (GE Healthcare) as 2nd antibodies. The blot was developed using either SuperSignalTM West Femto maximum sensitivity substrate or SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) according to the manufacturers’ instructions. Digital images were acquired with ChemiDoc™ Imaging Systems (Bio-Rad). Protein levels were quantified with Image Lab™ Software (Bio-Rad) and normalized against Actin or Lamin. Statistics was performed using two-way ANOVA.
3
Western Blot Analysis of DNA Damage Response
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Cells were harvested by trypsinization, and resuspended in urea lysis buffer (9 M urea, 150 mM β-mercaptoethanol, 75 mM Tris, pH 7.4). The lysate were sheared by sonication, centrifuged at 15000 rpm for 30 min, and the soluble fractions were collected. Protein concentration was determined using Quick StartTM Bradford 1x Dye Reagent (BioRad) and equal amounts of proteins were separated on 7.5% Tris-HCl SDS-polyacrylamid gels and transferred to Immobilon-P membrane (IPVH00010; EMD Millipore). The membrane was blocked in 5% milk in TBS with 0.02% Tween-20 and incubated with anti-ATM antibody (NB100–309; Novus), anti-ATM (phosphor S1981) antibody (ab81292; abcam), anti-MRE11 antibody (NB100–142; Novus), and anti-GAPDH antibody (#5174; Cell Signaling). ECLTM Anti-rabbit IgG, horseradish peroxidase linked whole antibody (NA934V; GE Healthcare) and ECLTM Anti-mouse IgG, horseradish peroxidase linked whole antibody (NA931V; GE Healthcare) were used as secondary antibodies. ECL western blotting substrate (Pierce) was used for detection by FluorChemTM E (Protein Simple).
4
Muscle Protein Extraction and Western Blot
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Proteins were extracted using boiling Laemmli buffer (2.5% SDS, 0.125 M Tris–HCl pH 6.8). Frozen pieces of muscle or C2C12 cells were mechanically dissociated in Laemmli buffer and incubated at 100 °C for 3 min. Primary antibodies used were anti-ErbB2 (1:1000, #sc-284), anti-ErbB3 (1:1000, #sc-285), anti-β-actin (1:4000, #sc-2228), anti-Murf1 (1:4000, #sc-32920) (Santa Cruz); anti-p-FoxO3a (Thr32) (1:1000, #9464), anti-FoxO3a (1:1000, #2497), anti-AKT (1:1000, #9272), anti-p-AKT (1:1000, #4051), anti-ERK (1:1000, #9102), anti-p-ERK (1:1000, #9106) (Cell Signalling); anti-Atrogin (1:3000, #ab168372, Abcam). The secondary antibodies used were ECLTM anti-rabbit IgG (1:40000, #NA934) and ECLTM anti-mouse IgG (1:40000, #NA931) (GE Healthcare). All the antibodies used detected a band of the predicted molecular weight. ImageJ was used for a quantitative analysis of the western blot bands obtained.
5
Western Blot Analysis of Protein Samples
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Protein samples were resolved by SDS-polyacrylamide gel electrophoresis and electrotransferred on a polyvinylidene difluoride membrane (Pall Corp., Port Washington, NY). The samples were then incubated with primary antibodies against NHE9 (1:800 dilution, Abcam, Cambridge, MA), GAPDH (1:5000 dilution, KangChen, Shanghai), β-actin (1:1000 dilution, Cell signaling), Caspase 3 (1:1000 dilution, Cell signaling), phosphor-Akt (1:1000 dilution, Cell signaling), phosphor-Src (Tyr416, 1:1000 dilution, Cell signaling), PARP (1:1000 dilution, Cell signaling), β-catenin (1:1000 dilution, BD), pGSK3β (1:1000 dilution, Cell signaling), Bcl-2 (1:1000 dilution, Cell signaling), Flag-tag (1:1000 dilution, Sigma), c-Myc (1:200 dilution, Santa), normal mouse IgG (1:200 dilution, Santa). The secondary antibodies used were ECLTM anti-mouse IgG (1:2500 dilution, GE healthcare) and ECLTM anti-rabbit IgG (1:2500 dilution, GE healthcare). The immunoreactive signals were detected with enhanced chemiluminescence kit (Amersham Biosciences, Uppsala, Sweden). The procedures followed were conducted in accordance with the manufacturer's instructions.
6
Immunoblotting for HCV Core Protein
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Primary antibodies were anti-HCV Core C7-50 (Santa Cruz Biotechnology, USA), anti-HA (MBL, Japan), anti-Flag (MBL), and anti-β-actin-HRP (horseradish peroxidase) (Protein Tech, China). The secondary antibodies were goat anti-rabbit IgG-HRP (Protein Tech) and goat anti-mouse IgG-HRP (Protein Tech) or anti-mouse IgG-Alexa Fluors (Life Technologies). ECLTM anti-mouse IgG and a HRP-linked whole antibody (GE Healthcare, UK) were used for the FFU assay.
7
Antibody Characterization for Cell Signaling
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The antibodies used were purchased commercially from the following manufacturers: mouse monoclonal anti-FLAG (SIGMA-ALDRICH), rabbit polyclonal anti-FLAG (SIG-MA-ALDRICH), mouse monoclonal anti-SNX1 (sc-136247; Santa Cruz), mouse mono-clonal anti-SNX2 (sc-136072; Santa Cruz), rabbit polyclonal anti-SNX5 (Santa Cruz), rabbit polyclonal anti-SNX6 (Santa Cruz), rabbit polyclonal anti-GAPDH (Santa Cruz), mouse monoclonal anti-EEA1 (MBL), mouse monoclonal anti-TGN46 (SIGMA-ALDRICH), ECLTM anti-mouse IgG, horseradish peroxidase linked whole antibody (GE Healthcare), ECLTM anti-rabbit IgG, horseradish peroxidase linked whole antibody (GE Healthcare), Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen), Alexa Fluor 568 goat anti-mouse IgG (Invitrogen). Anti-CI-M6PR rabbit polyclonal was kindly provided by Dr. Jack Rohrer (Zurich University of Applied Sciences). Human recombinant EGF and FITC-conjugated dextran 70 k were obtained from Wako pure chemicals industries and Molecular probe, respectively.
8
Quantification of Insulin Receptor Protein
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Protein was extracted from liver and the four skeletal muscles and the protein concentration in the supernatant was determined using a bicinchoninic acid assay (BCA kit; Pierce, Rockford, IL). Western Blotting was performed as described previously 32 using a primary antibody to the insulin receptor (InsR, subunit β (Insulin Rβ (C-19): (1:200; sc-711, Santa Cruz Biotechnology, inc, Delawere Avenue, Santa Cruz, CA, USA) and to β-actin (1:10,000; sc-47778, Santa Cruz Biotechnology, inc, Delawere Avenue, Santa Cruz, CA, USA) to evaluate protein loading. Horseradish peroxidase-conjugated secondary antibodies to the host species were used as appropriate (1:5,000-1:10,000; ECL TM Anti-rabbit IgG and ECL TM Anti-mouse IgG; GE Healthcare Life Sciences, Little Chalfont, Bucks, UK). Protein abundance was detected using enhanced chemiluminescence reaction (ECL plus kit, Amersham Hiperfilm Bioscience, UK) and X-ray film (Fuji FPM100A processor) and analysed using the ImageJ program (ImageJ, Software, National Institute of Health, Bethesda, MD; http://rsb.info.nih.gov/ij). The depicted bands had the expected molecular weights.
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