Samples were homogenized in lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 0.2% Triton X-100, 0.3% NP-40, 0.1 mmol/L PMSF, 25 mmol/L NaF). Proteins were separated by 10% SDS-PAGE under reducing conditions, then blotted onto nitrocellulose membranes. Membrane blockade was accomplished with 5% defatted milk in TBS-T (0.05 mol/L Tris, 0.15 mol/L NaCl, 0.05% Tween 20, pH 7.8). Thereafter, membranes were overnight probed at 4°C with primary antibodies in the same blocking solution or 5% BSA in TBS-T and then incubated with secondary HRP-conjugated antibodies for 1 h at room temperature. The following primary antibodies were used to detect specific proteins of interest: rabbit polyclonal anti-p-STAT1 (Tyr701) (Invitrogen, 44-376G), p-TYK2 (pTyr1054) (Origene, TA333304), and p-IKKε (Ser172) (Sigma Aldrich, 06-1340); rabbit monoclonal anti-p-TBK1/NAK (S172) (D52C2) XP® (Cell Signaling Technology, 1,675,483), TBK1/NAK (E8I3G) (Cell Signaling Technology, 38,066), IKKε (D61F9) XP® (Cell Signalling Technology, 3416), pIRF3 (Ser396) (4D4G) (Cell Signalling Technology, 4,947) and IRF-3 (D83B9) (Cell Signalling Technology, 4,302); monoclonal mouse anti-pIKBα (Santa Cruz, sc-8404). Anti-α-Tubulin (Sigma-Aldrich, MAB374) and anti-GAPDH (Millipore, MAB374) were used to assess protein loading homogeneity.
Mab374
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, France, Canada, Macao, Switzerland
MAB374 is a laboratory equipment product manufactured by Merck Group. It is a high-precision instrument designed for specific scientific applications. The core function of MAB374 is to perform accurate measurements and analysis within controlled experimental settings. Further details on its intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (16 mentions)
Gapdh, by Merck Group (16 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Bca protein assay, by Thermo Fisher Scientific (8 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (8 mentions)
Protease inhibitor, by Roche (8 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Ab1791, by Abcam (7 mentions)
Anti gapdh, by Merck Group (7 mentions)
Odyssey infrared imaging system, by LI COR (7 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (6 mentions)
Ripa buffer, by Merck Group (6 mentions)
Image lab software, by Bio-Rad (6 mentions)
Clarity western ecl substrate, by Bio-Rad (6 mentions)
Nitrocellulose membrane, by Bio-Rad (6 mentions)
Chemidoc mp imaging system, by Bio-Rad (6 mentions)
Bca assay, by Thermo Fisher Scientific (6 mentions)
Odyssey imaging system, by LI COR (5 mentions)
342 protocols using mab374
1
Immunoblotting Analysis of Signaling Proteins
2
Retinal Immunostaining and Western Blot
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Whole-mounted retinas were prepared for immunostaining and retinal protein samples for Western blot analysis as described previously (Liu et al., 2007 (link); Yoshida et al., 2011 (link); Puyang et al., 2016 (link); Feng et al., 2016 (link)). Primary antibodies for immunostaining included rabbit anti–green fluorescent protein (anti-GFP; 1:1000; Thermo Fisher Scientific, A-6455), mouse anti–Brn-3a (1:400; EMD Millipore, MAB1585), goat anti–Brn-3b (1:1000; Santa Cruz Biotechnology, sc-6026), mouse anti-TH (tyrosine hydroxylase, 1:400, EMD Millipore, MAB318), choline acetyltransferase (ChAT; 1:200; EMD Millipore, MAB305; Liu et al., 2007 (link)), BETA3 (also known as BhlhB5, 1:1000; Santa Cruz Biotechnology, sc-6045; Feng et al., 2006 (link)), cocaine- and amphetamine-regulated transcript (CART; 1:2500, Phoenix Pharmaceuticals), SMI-32 (neurofilament H non-phosphorylated, 1:1000, Covance; Feng et al., 2013 (link)), rabbit anti-BDNF (1:400; EMD Millipore, Ab1779SP; Feng et al., 2016 (link)), and mouse anti-GAPDH (1:2000, EMD Millipore, MAB374; Yoshida et al., 2011 (link)). Alexa Fluor–conjugated secondary antibodies were used (1:1000; Invitrogen), and images were captured with a Zeiss Pascal confocal microscope. Western blot analysis was performed using rabbit anti-BDNF (1:400; EMD Millipore, Ab1779SP) and mouse anti-GAPDH (1:2000, EMD Millipore, MAB374; Feng et al., 2016 (link)).
3
Western Blot Analysis of Trbp Protein
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Protein lysate samples were prepared from heart tissues in RIPA buffer with proteinase inhibitors. Lysate samples (15–20 µg total protein for each) were separated by 12% SDS-PAGE, and electrophoretically transferred to PVDF membranes. Trbp protein was probed with Rabbit or Mouse anti-Trbp antibodies (AbCam, #42018, or Thermo Scientific, #LF-MA0209, 1:1,000). Flag-tagged proteins were probed with Rabbit anti-Flag M2 (Sigma, #F7425, 1:2,000). Gapdh, or β-tubulin used as loading controls, were probed with mouse anti-Gapdh (EMD Millipore, MAB374, 1:10,000) or mouse anti- β-Tubulin(Sigma, #T8328, 1:5,000) antibodies, respectively. Protein bands were visualized with Oddessay image system.
4
Quantification of PPARγ Expression
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Adipose tissue was homogenized in ice-cold RIPA buffer with protease inhibitors (Protease inhibitor Cocktail Set III, Calbiochem, La Jolla, CA). Protein concentration was quantified using a BCA protein assay (Thermo Fisher Scientific, Hanover Park, IL, USA). For immunoblots, 30 µg of protein sample was separated by 10% SDS-PAGE and transferred to a PVDF membrane (MilliporeSigma, Burlington, MA, USA). Membranes were blocked in 5% nonfat dry milk (w/v) and incubated with a primary antibody against PPARγ (Santa Cruz Biotechnology, sc-7273, Santa Cruz, CA, USA) or GAPDH (MAB374, Sigma-Aldrich, St. Louis, MO, USA), followed by IgG-horseradish peroxidase-conjugated secondary antibody. Blots were developed using Amersham ECL Prime Detection Reagent (VWR, West Chester, PA, USA).
5
Western Blot Analysis of Primary CD4+ T Cells
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Primary CD4+ T cells were lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with 1x protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Lysates were incubated with DTT and NuPAGE LDS sample buffer (Invitrogen). Lysates were run on 12% Criterion TGX gels (BioRad) and transferred to Amersham Hybond polyvinylidene difluoride (PVDF) blotting membranes (Cytiva). Western blots were blocked in 5% BSA in TBS 0.1% Tween and washed in TBS 0.2% Tween. Western blots were incubated with secondary antibody in 5% milk in TBS 0.1% Tween. Western blot chemiluminescence was detected with SuperSignalTM West Femto Substrate (Thermo Scientific). Imaging of the Western blot bands was performed using AlphaView software (ProteinSimple). The following antibodies were used for immunoblots: α-ISG15 (clone F9, 1:500, Santa Cruz Biotech, Cat# sc-166755), α-MX1 (1:1000, Abcam, ab95926), α-GAPDH (clone 6C5, 1:10,000, Sigma Aldrich, MAB374).
6
Western Blot Analysis for Protein Detection
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Western blot analysis was performed as previously described [23 (link)]. In brief, lysates samples were prepared from tissues or cultured cells in RIPA-based buffer, separated by SDS-PAGE gel, and electrophoretically transferred to PVDF membranes. The membrane was probed with a mouse anti-Trbp antibody (Thermo Scientific, #LF-MA0209) and the mouse MF20 antibody. Gapdh, or β-tubulin was used as loading controls (mouse anti-Gapdh EMD Millipore, MAB374 or mouse anti-β-tubulin,Sigma, T8328, respectively). Protein bands were visualized with Odyssay image system (LI-COR).
7
Western Blot Analysis of Pancreatic Transcription Factors
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Cells were collected and lysed with RIPA Buffer (Wako) containing 1% protease inhibitor cocktail (Sigma-Aldrich). The lysate was mixed with Sample Buffer Solution with 2-mercaptoethanol (Nacalai Tesque), incubated for 3 min at 95 °C, loaded into a 4–20% Mini-protein TGX precast gel (Bio-Rad) and transferred to a PVDF membrane using a Trans-Blot Turbo Transfer System (Bio-Rad) after electrophoresis. Then, the membrane was blocked with 5% skim milk in Tris-based saline with Tween 20 (0.1% TBS-T) buffer and incubated with the following primary antibodies diluted in blocking solution overnight at 4 °C: rabbit anti-HHEX (R&D Systems, MAB83771-100, 1:200), goat anti-PDX1 (R&D Systems, AF2419, 1:5000), rabbit anti-NKX6.1 (Cell Signaling, 54551, 1:1000) and mouse anti-GAPDH (Sigma-Aldrich, MAB374, 1:300). After washing with TBS-T, the membrane was incubated with the following secondary antibodies diluted in blocking solution for an hour at room temperature: horseradish peroxidase (HRP) conjugated sheep anti-mouse (Cytiva, NA931VS, 1:3000), HRP conjugated donkey anti-rabbit (Cytiva, NA934VS, 1:3000) and HRP conjugated donkey anti-goat (Abcam, ab6885, 1:5000). After washing with TBS-T, the membrane was incubated with ECL Western Blotting Detection Reagent (Amersham) for 5 min at room temperature. The protein bands were visualized using an ImageQuant 800 (Cytiva).
8
Comprehensive Antibody Characterization Protocol
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Monoclonal antibodies used included: AT270 (MN1050, Thermo Fisher Scientific, RRID: AB_223651), AT180 (MN1040, Thermo Fisher Scientific, RRID: AB_223649), HT7 (MN1000, Thermo Fisher Scientific, RRID: AB_2314654), Tau5 (AHB0042, Thermo Fisher Scientific, RRID: AB_2536235), MAb359 (from Dr. Li Gan), anti-GAPDH (MAB374, Sigma-Aldrich, RRID: AB_2107445), anti-FLAG (F1804, Sigma-Aldrich, RRID: AB_262044), AT8 (MN1020, Thermo Fisher Scientific, RRID: AB223647), anti-SV2 (University of Iowa DSHB, RRID: AB_2315387), anti-PICK1 (75–040, Antibodies Inc, RRID: AB_2164544), anti-KIBRA (sc-133374, Santa Cruz Biotechnology, RRID:AB_2216359). Polyclonal antibodies used included: anti-PKMζ (from Dr. Todd C. Sacktor), anti-PSD95 (2507, Cell Signaling Technology, RRID: AB_561221), anti-HA (H6908, Sigma-Aldrich, AB_260070), GFP (A-21311, Thermo Fisher Scientific, RRID: AB_221477), anti-GluA1 (ABN241, Millipore, RRID: AB_2721164)
9
Antibody-Based Protein Expression Analysis
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Primary antibodies used in this study were ATF6 (100 ng/mL, NBP1‐40256, Novus Biologicals), CHOP (C/EBP‐homologous protein) (1:10.000, 2895, Cell Signaling, Danvers, MA), GRP78 (70 ng/mL, 11587‐1‐AP, Proteintech, Rosemont, IL), IRE1α phosphorylated at Ser724 (200 ng/mL, MBS9610512, MyBioSource, San Diego, CA), IRE1α (40 ng/mL, sc‐390960, Santa Cruz Biotechnology, Dallas, TX), eIF2α phosphorylated at Ser51 (1:10.000, 3398, Cell Signaling), eIF2α (1:10.000, 2103, Cell Signaling), GAPDH (100 ng/mL, MAB374; Sigma‐Aldrich, St. Louis, MO), and β‐actin (6.1 ng/mL, 4970; Cell Signaling Technology).
10
Subcellular Fractionation and Western Blot Analysis
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For Western blot analysis, C2C12 cells and muscle tissues were homogenized with Mammalian Cell Lysis Buffer (Sigma Aldrich) and Cell Lysis Regent for mammalian tissue (Sigma Aldrich), respectively. For subcellular fractionation, skeletal muscle tissue (quadriceps) was homogenized by a polytron homogenizer in 40 volumes (w/v) of buffer A (50mM Tris-HCl, ph 7.5, 1 mM DTT, 1 mM EDTA) containing protease inhibitor cocktail (Nacalai tesque, Tokyo, Japan). The homogenates were then centrifuged for 30 min at 100,000 x g to obtain the membrane pellet and cytosol fractions. The pellet fraction was resuspended and homogenized in 40 volumes of buffer A containing 1% NP40 (Sigma Aldrich).
Western blot analysis was performed as described previously [13 (link),14 (link)]. The following antibodies were used: anti-SIRT1 (1:1000 dilution; ab110304, Abcam), anti-dystrophin (1:1000 dilution; ab15277, Abcam), anti-caveolin 1 (1:1000 dilution; ab2910, Abcam), anti-caveolin 3 (1:1000 dilution; ab30750, Abcam), anti-nNOS (1:200 dilution; A-11, Santa Cruz), anti-Histone H3 (1:2000 dilution; ab1791, Abcam), anti-Histone H3 acetyl K9 (1:10,000 dilution; ab4441, Abcam), anti-GAPDH (1:2000 dilution; MAB374, Sigma Aldrich) and anti-α-tubulin (1:2000 dilution; T5168, Sigma Aldrich).
Western blot analysis was performed as described previously [13 (link),14 (link)]. The following antibodies were used: anti-SIRT1 (1:1000 dilution; ab110304, Abcam), anti-dystrophin (1:1000 dilution; ab15277, Abcam), anti-caveolin 1 (1:1000 dilution; ab2910, Abcam), anti-caveolin 3 (1:1000 dilution; ab30750, Abcam), anti-nNOS (1:200 dilution; A-11, Santa Cruz), anti-Histone H3 (1:2000 dilution; ab1791, Abcam), anti-Histone H3 acetyl K9 (1:10,000 dilution; ab4441, Abcam), anti-GAPDH (1:2000 dilution; MAB374, Sigma Aldrich) and anti-α-tubulin (1:2000 dilution; T5168, Sigma Aldrich).
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