Total lysates from mid-log-phase cells were harvested and lysed in 1 mL of ice-cold lysis buffer (300 mM NaOH, 1% β-mercaptoethanol). Proteins were precipitated through the addition of TCA to 7.5% and resuspended in HU buffer (8 M Urea, 5% SDS, 200 mM Tris pH 6.8, 100 mM DTT, 1 mM ethylenediaminetetraacetic acid (EDTA), bromophenol blue) using a volume that was normalized to the amount of cells harvested. Proteins were separated by SDS–PAGE and analyzed by immunoblotting. The following antibodies were used: rabbit anti phospho- eIF2α (Ser51) (Cell Signaling Technology, cat# 9721S), rabbit anti-Gcn4 (from Thomas Dever lab, 1:2000 v/v dilution), rabbit anti Gcn1 (from Alan Hinnebusch lab, 1:2000 v/v dilution), mouse anti-ubiquitin HRP (Santa Cruz, Cat#: sc8017; 1:2,000 v/v dilution), mouse anti-FLAG (Sigma; Cat#: F1804; 1:5,000 v/v dilution), rabbit anti-uS4 (Rps9) (Abcam, Cat#:ab117861; 1:5,000 v/v dilution), and rabbit anti-eRF1(Eyler et al., 2013 (link)) (1:2000 dilution). Secondary antibodies of goat anti mouse IgG HRP (Thermo Scientific, Cat#: 31430) and goat anti rabbit IgG HRP (Thermo Scientific, Cat#: 31460) were used at (1:10,000 v/v dilution). Densitometry analysis was conducted using Image Lab (Bio-Rad Laboratories). Unless otherwise stated, relative signal of the protein of interest was obtained by normalizing to that of PGK1.
Goat anti mouse igg hrp
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat anti-mouse IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is used to detect and quantify mouse immunoglobulin G (IgG) in various immunoassays and immunohistochemical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg hrp, by Thermo Fisher Scientific (37 mentions)
β actin, by Merck Group (4 mentions)
Ecl kit, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Bradford assay, by Bio-Rad (3 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Goat anti rabbit, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Mouse anti flag, by Merck Group (2 mentions)
Dylight 549 goat anti mouse igg, by Abbkine (2 mentions)
Dylight 488 goat anti mouse igg, by Abbkine (2 mentions)
Goat anti rabbit igg hrp, by Abcam (2 mentions)
Imager 680, by Cytiva (2 mentions)
Goat anti guinea pig igg hrp, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Tmb substrate, by Thermo Fisher Scientific (2 mentions)
Ecl solution, by Cyanagen (2 mentions)
107 protocols using goat anti mouse igg hrp
1
Analyzing Stress Response Signaling
2
Western Blot Analysis of Epigenetic Regulators
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15 µL protein sample was loaded into 10% polyacrylamide gel. Electrophoresis and transfer were performed using Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, USA). The membrane was blocked for 1 h with 5% BSA in TBST buffer (Tris: 20 mM, NaCl: 150 mM, Tween® 20 detergent: 0.1% (w/v)). Immunoblot analysis was conducted with the following antibodies: β-Actin (Sigma, A2228, 1:1000); H3K27Me3 (Abcam, ab6002, 1:1000); Histone 3 (Abcam, ab1791, 1:1000); anti-Brg1 (Abcam, ab4081, 1:1000); Ezh2 (Cell Signaling, D2C9 #5246, 1:1000); REST/NRSF (Millipore, 17-641, 1:1000); GAPDH‐HRP (Prosci, HRP‐60004, 1:1000); goat anti‐mouse IgG HRP (ThermoFisher Scientific, 62‐6520, 1:2500), and goat anti‐rabbit IgG HRP (ThermoFisher Scientific, 65‐6120, 1:2500). Protein bands were developed with Pierce™ ECL Western Blotting Substrate (ThermoFisher Scientific, 32209) and were imaged using the ChemiDoc MP Imaging system (Bio‐Rad, Hercules, CA, USA). Relative band densities were analyzed using ImageJ.
3
Western Blot Analysis of MMP-2, NF-κB, and p38
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Western blot analysis was performed as previously described [16 (link)]. Briefly, whole-cell lysates were prepared in lysis buffer (Cell Signaling Technology, MA, USA). Samples containing 30 μg of protein were resolved by electrophoresis gels and transferred to a nitrocellulose membrane. After blocking for 1h with 5% skim milk in PBST, membranes were incubated overnight at 4 °C with anti-MMP-2 (MAB902, R&D Systems; Minneapolis, MN, USA), NF-κB p65 (sc-8008, Santa Cruz Biotechnology, Dallas, TX, USA), p38 (D13E1-Cell Signaling Technology, MA, USA), phospho–p38 (D3F9-Cell Signaling Technology, MA, USA), and anti-β-actin (Millipore Corporation, Billerica, MA, USA) primary antibodies. Membranes were then probed with goat anti-mouse IgG-HRP (31430, Thermo Scientific) secondary antibody at room temperature (RT) for 1 h. Detection of the immunoreactive bands was performed using the Clarity Western ECL Substrate (BioRad). β-actin served as a loading control. Densitometric analysis was performed using Image J.
4
Co-Immunoprecipitation Experiments with Flag-tagged Proteins
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Co-IP experiments were performed as previously described (Shyian et al., 2020 (link)) with the following modifications. OD600 ~ 0.9 cell cultures were used to prepare 1 ml of lysate. Cells were broken with glass beads in a Precellys Evolution homogenizer. 20 µl of anti-Flag M2 gel (Sigma) per 1 ml of lysate were used. Proteins were eluted by boiling at 95 °C for 10 min in 30 µl of 2 x SDS-PAGE buffer (100 mM Tris pH 8, 4% SDS, 10% glycerol, 0.2% bromophenol blue). Total proteins were isolated as previously described (Kushnirov, 2000 (link)). Proteins were separated on 8% PAGE gels, transferred onto Hybond P 0.22 PVDF membrane (GE Healthcare), stained with Ponceau S (Amresco), and blocked in 5% BSA. Anti-HA-HRP antibodies (clone 3F10, Sigma) at 1:5000, anti-FLAG M2 antibodies (Sigma) at 1:5000, goat anti-mouse IgG-HRP (62–6520, Thermo Fisher Scientific) at 1:5000 and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) were used for protein detection.
RNA Co-IP experiments were performed as described in Shepelev et al., 2020 (link).
RNA Co-IP experiments were performed as described in Shepelev et al., 2020 (link).
5
Immunofluorescence Antibody Staining Protocol
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The mouse monoclonal anti-RSV antibody was kindly provided by Prof. Jianxiang Wu (Zhejiang University). The rabbit anti-RSV antibody was the kind gift of Yan Huo (Chinese Academy of Sciences). An anti-LsST6 monoclonal antibody against the LsST6 peptide SKGDHNTEAALP was produced by Abmart (Shanghai, China). The following antibodies were obtained from the sources indicated: mouse monoclonal anti-Myc tag (cat. 66004, Proteintech), rabbit polyclonal anti-His tag (cat. 2365, Cell Signaling Technology), Dylight 488 goat anti-rabbit IgG (cat. A23220, Abbkine), Dylight 488 goat anti-mouse IgG (cat. A23210, Abbkine), Dylight 549 goat-anti-mouse IgG (cat. A23310, Abbkine), goat anti-mouse IgG+HRP (cat. 32430, Thermo), goat anti-rabbit IgG+HRP (cat. 32460, Thermo). Alexa Fluor 633 phalloidin was obtained from Invitrogen and 4’,6-diamidino-2-phenylindole (DAPI) was purchased from Sigma.
6
ELISA-Based Anti-RSV IgG Quantification
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Sera were evaluated for anti-RSV IgG levels as described (51 (link)). Briefly, high-binding ELISA plates (Corning, Corning, NY) were coated with 5 µg/mL RSV A2 or B1 lysate overnight at 4°C. The next day, wells were washed 3x with KPL wash buffer (1x KPL in distilled water (diH2O) (SeraCare, Milford, MA) and blocked with Blotto (5% non-fat dry milk) overnight at 4°C. Blotto was removed and sera (in 3-fold dilutions starting at 1:50) was diluted in Blotto and added to wells overnight at 4°C. Wells were washed 3x with KPL wash buffer and 2° goat-anti-mouse IgG-HRP (ThermoFisher, Waltham, MA), or secondary subtype IgG1 or IgG2a antibodies (Southern Biotech, Birmingham, AL) were added. Plates were incubated overnight at 4°C, washed 3x with KPL wash buffer, and developed with 1-Step™ Ultra 3,3’,5,5’-tetramethylbenzidine (TMB; ThermoFisher) for 20 min, and stopped with Stop Solution (ThermoFisher), then read immediately using a BioTek plate reader (BioTek, Winooski, VT) at OD450.
7
Immunofluorescence and Western Blotting Antibodies
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Primary antibodies used for immunofluorescence microscopy and Western Blotting were obtained from Cell Signaling Technology and included: Beta-actin (#3700 and #4970), phospho-p70s6k T389 (#9234), NF kappa B p65 (#8242S), beta-tubulin (#2128S), Caspase-3 (#14220), Caspase-1 (#3866), Phospho-MLKL (#91689). Secondary antibodies used to detect the primary antibodies for Western Blotting were obtained from Thermo Fisher Scientific, and included goat anti-rabbit and goat anti-mouse IgG-HRP (#31460 and #31430). Secondary antibodies used for immunofluorescence microscopy were obtained from Molecular Probes (Life Technologies), and included goat anti-rabbit IgG AlexaFluor488 and goat anti-mouse IgG AlexaFluor594.
8
Phospho-RTK Array for Signaling Pathways
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The human Phospho-RTK Array Kit was obtained from R&D Systems. Phosphorylserine (OPS), sphingomyelin, and ADAM10 inhibitor (GI254023X) were from Sigma-Aldrich. Erlotinib Hydrochloride, a broad spectrum matrix metalloprotease (MMP) inhibitor GM6001, and ADAM17 inhibitor TAPI-1 were from Selleck. ADAM17/10 inhibitor (GW280264X) was from TargetMoI. EGFR-blocking antibody Cetuximab, ceramidase inhibitor Ceranib1, and ceramide kinase inhibitor NVP231 were from MCE. Annexin V-FITC from BD Bioscience. Anti-GAPDH, anti-EGFR, anti-phosphoEGFR, anti-ERK1/2, anti-phosphoERK1/2, and anti-TACE (ADAM17) were from Cell Signaling Technology (1:1,000). Goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP were from Thermo Fisher Scientific (1:4,000). Blocking and neutralizing antibodies against human epiregulin, HB-EGF, and TGF-α antibodies were from R&D Systems (0.8 µg/mL). Additional antibodies used for flow cytometry were as follows: Brilliant Violet 605 anti-mouse CD11c (N418), FITC anti-mouse CD45 (30-F1), PerCP/Cyanine5.5 anti-mouse Ly-6G/Ly-6C (Gr-1) (RB6-8C5), PE anti-mouse F4/80 (BM8), and APC-anti-mouse/human. CD11b (M1/70) was from Biolegend (1:100).
9
Protein Extraction and Immunoblotting Protocol
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Total protein was extracted from bone marrow samples and cells using RIPA lysis buffer (Beyotime Biotechnology, China) with 1× Thermo Scientific™ Halt™ Protease Inhibitor Cocktail (Thermo Fisher, USA) according to the manufacturer’s instructions. For tumor samples, the tumor cells were first homogenized from the tumor tissues by a freezing grinder (LUKYM-II, China), and then, the total protein was extracted from the tumor cells.
For Immunoblotting, all protein samples were suspended in 5x loading buffer and then denatured for 5 min at 100 °C, separated via SDS-PAGE, transferred to PVDF membranes, and blotted. The antibodies are as fellow: MLL1 (BETHYL, A300-086 A-3); LC3B (Sigma, L7543); LAMP5(Sigma, A78501); β-tubulin (Invitrogen, 32-2600); β-actin (Sigma, A2228); GAPDH (Proteintech, 10494-1-AP); DOT1L (CST, 90,878 S); H3(Abcam, Ab1791); H3K79me2(Abcam, Ab3594); H3K79me3(Abcam, Ab2621); IgG (CST, 3900); Goat Anti-Mouse IgG HRP (Thermo Fisher, H10007); Goat Anti-Rabbit IgG HRP (Thermo Fisher, A18903); Goat Anti-Rabbit IgG H&L DyLight® 594(Abcam, ab96885);Goat Anti-Rabbit IgG H&L DyLight® 647 (Abcam, ab150115).
For Immunoblotting, all protein samples were suspended in 5x loading buffer and then denatured for 5 min at 100 °C, separated via SDS-PAGE, transferred to PVDF membranes, and blotted. The antibodies are as fellow: MLL1 (BETHYL, A300-086 A-3); LC3B (Sigma, L7543); LAMP5(Sigma, A78501); β-tubulin (Invitrogen, 32-2600); β-actin (Sigma, A2228); GAPDH (Proteintech, 10494-1-AP); DOT1L (CST, 90,878 S); H3(Abcam, Ab1791); H3K79me2(Abcam, Ab3594); H3K79me3(Abcam, Ab2621); IgG (CST, 3900); Goat Anti-Mouse IgG HRP (Thermo Fisher, H10007); Goat Anti-Rabbit IgG HRP (Thermo Fisher, A18903); Goat Anti-Rabbit IgG H&L DyLight® 594(Abcam, ab96885);Goat Anti-Rabbit IgG H&L DyLight® 647 (Abcam, ab150115).
10
Western Blot Analysis of Cellular Proteins
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3*106 cells or tumor tissue was lysed in cell lysis buffer (Cell Signaling Technologies). Protein concentrations were determined using Bradford protein assay (Bio-rad) and normalized to 1 µg/µL, followed by sample reduction and denaturation in 1x Laemmli sample buffer (Bio-rad) containing 2-mercaptoethanol. Protein lysates (10-15uL) were separated on Criterion TGX Tris-Glycine eXtended (TGX) precast gels (Bio-rad), transferred to PVDF membranes using the Trans-Blot Turbo system (Bio-rad), and incubated overnight with primary RPL5 (Abcam), α-Tubulin (Sigma Aldrich), Actin (Sigma Aldrich), phospho-CDC2/CDK1 (Tyr15), c-MYC, MDM2 or p53 (all from Cell Signaling) antibody, washed, and incubated for 1 hour with secondary Goat Anti-Mouse IgG-HRP or Goat Anti-Rabbit IgG-HRP antibodies (Thermo Fisher). Protein bands were visualized using chemiluminescent chemistry on an Azure C600 (Azure Biosystems). Quantification was performed using LI-COR Image Studio Lite software version 5.2.
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