Cells in 35 mm culture plates were lysed with 90 µL of SDS buffer (2% SDS, 10% glycerol, and 62.5 mM Tris-HCl, pH 6.8). Protein samples, boiled in Laemmli sample buffer with 100 mM DTT for 5 min, were separated by 10% SDS-PAGE (40 µg per lane) and transferred to a nitrocellulose membrane (Protran, PerkinElmer, Shelton, CT) using a semi-dry transfer system (BioRad). Blocked membranes were incubated overnight at 4 °C with monoclonal mouse anti human PCNA primary antibody, PC10, (Santa Cruz Biotechnology, Santa Cruz, CA), rinsed, and then incubated with horseradish peroxidase conjugated goat anti mouse secondary antibody (BioRad) for 1 hour at room temperature. Protein bands were detected by Super Signal West Pico chemiluminescent substrate (Pierce, Rockford, IL) using X-ray film. The apparent molecular weights of PCNA forms induced by Nic-Cl were estimated by comparison to molecular weight markers.
Horseradish peroxidase conjugated goat anti mouse secondary antibody
Manufactured by Bio-Rad
Sourced in United States
Horseradish peroxidase conjugated goat anti mouse secondary antibody is a laboratory reagent used for the detection and quantification of mouse primary antibodies in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry. It consists of a goat-derived secondary antibody that is chemically conjugated to the enzyme horseradish peroxidase, which catalyzes a colorimetric or chemiluminescent reaction for signal amplification and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Bio-Rad (4 mentions)
Horseradish peroxidase conjugated anti flag antibody, by Merck Group (3 mentions)
Horseradish peroxidase conjugated anti ha antibody, by Roche (2 mentions)
Protran, by PerkinElmer (1 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
Immobilon p pvdf, by Merck Group (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Immun blot, by Bio-Rad (1 mentions)
Nupage tris acetate sds running buffer, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Rc dctm protein assay, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
V5 antibody, by Thermo Fisher Scientific (1 mentions)
P53 antibody do 1, by Santa Cruz Biotechnology (1 mentions)
Onx 0914, by Apexbio (1 mentions)
Goat anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
14 protocols using horseradish peroxidase conjugated goat anti mouse secondary antibody
1
Western Blot Analysis of PCNA in Nic-Cl Treated Cells
2
Immunoblotting for Activation-Induced Cytidine Deaminase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected from a 5 ml culture sample (14 000 rpm; 1 min, 4°C), treated with 5% trichloroacetic acid (Sigma-Aldrich) and processed as described (16 (link)). Membranes (Immobilon-P PVDF; Millipore) were incubated with mouse monoclonal anti-AID antibody (1:1000; Cosmo Bio Co., Cat # BRS-APC004AM) or with mouse monoclonal anti-Pgk1 antibody (1:5000; Life Technologies, Cat # 459250). Following incubation with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:10 000; BioRad, Cat # 170–6516) the proteins were visualized using ECL chemiluminescence solution (GE Healthcare) and radiography (GE Healthcare).
3
Western Blot Analysis of C. albicans Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At mid-log phase, 10 ml cultures of C. albicans were collected by centrifugation at 3,000g for 10 min at 4°C. Cell pellets were washed with 1 ml ice-cold water then flash frozen in liquid N2. Whole-cell lysates were prepared by mild alkaline lysis, trichloroacetic acid precipitation, and solubilized in SDS/urea loading buffer as previously described, other than omission of bromophenol blue dye. Protein concentration was roughly normalized by absorbance at 280 nm. Lysates were separated in NuPAGE 3–8% acrylamide Tris-acetate gels using NuPAGE Tris-acetate SDS running buffer (Thermo Fisher Scientific). Proteins were transferred to PVDF membranes (Bio-Rad Immun-Blot, 0.2 μm). Membranes were blocked with 3% (wt/vol) bovine serum albumin in Tris-buffered saline with 0.5% (vol/vol) Tween-20 (TBS-T). Membranes were probed with mouse–anti-His5 monoclonal primary antibody (QIAGEN; 1:3,000) and horseradish peroxidase-conjugated goat-anti mouse secondary antibody (Bio-Rad; 1:5,000). Chemiluminescence images were acquired on a Chemidoc Touch imaging system (Bio-Rad) using Clarity western ECL substrate (Bio-Rad).
4
NPAS3 Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nitrofen and control lungs were washed and protein was extracted using lysis buffer: 10 mM Tris-HCl (pH 6.8), 5 lM b-glycerophosphate, 20 lM EDTA, 5 % SDS, a protease inhibitor cocktail tablet and phosphatase inhibitors (1 mM sodium orthovanadate, 2 mM EGTA, 10 mM sodium pyrophosphate, 30 mM sodium chloride). Protein concentration was determined using RC DC TM Protein Assay (Bio-Rad). Fifteen lg of total proteins were reduced with b-mercaptoethanol, size fractionated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). NPAS 3 was detected with an NPAS 3 antibody (ProSci incorporated) using a 1:1000 dilution and a horseradish peroxidase-conjugated goat-anti-rabbit secondary antibody (Bio-Rad) in a 1:5000 dilution. For loading control, blots were probed with anti-GAPDH (Abcam, MA, USA) in a 1:10000 dilution, using a horseradish peroxidase-conjugated goat-anti-mouse secondary antibody in a 1:6000 dilution (Bio-Rad). Exposed films were scanned and densitometry was performed using ImageJ software (Wayne Rasband, NIH, USA).
5
Purification of Toxic Bacterial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
Escherichia coli BL21 (DE3) strains carrying pET28b-based plasmids were grown in LB supplemented with kanamycin and induced with 0.2 mM IPTG for 12 h at 25°C. Specifically, in order to purify toxin HepT and HepT/MntA variants that are toxic, stationary cells (OD600 ∼ 1.0) were induced with 0.5 mM IPTG for only 2 h at 37°C (longer induction periods and IPTG addition at inoculation resulted in too much toxicity). Cells expressing the protein of interest were collected, and the His-tagged proteins were purified following the method described in a previous study (21 (link)). For the western blot assay, protein samples were transferred to a PVDF membrane (Millipore, Bedford, MA, USA), and performed with primary antibodies raised against a His-tag (Cell Signaling Technology, Danvers, MA, USA) and horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (Bio-Rad, Richmond, CA, USA).
+ Expand
Escherichia coli BL21 (DE3) strains carrying pET28b-based plasmids were grown in LB supplemented with kanamycin and induced with 0.2 mM IPTG for 12 h at 25°C. Specifically, in order to purify toxin HepT and HepT/MntA variants that are toxic, stationary cells (OD600 ∼ 1.0) were induced with 0.5 mM IPTG for only 2 h at 37°C (longer induction periods and IPTG addition at inoculation resulted in too much toxicity). Cells expressing the protein of interest were collected, and the His-tagged proteins were purified following the method described in a previous study (21 (link)). For the western blot assay, protein samples were transferred to a PVDF membrane (Millipore, Bedford, MA, USA), and performed with primary antibodies raised against a His-tag (Cell Signaling Technology, Danvers, MA, USA) and horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (Bio-Rad, Richmond, CA, USA).
6
Lung Cancer Cell Line Cultivation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
H1299, H441, A549, and H460 [American Type Culture Collection (ATCC), Manassas, VA, USA] were cultured in RPMI-1640 medium that was supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere of 5% CO2 and 95% air. C-Met, PARP, and cleaved caspase 3 antibodies were from Cell Signaling Technology (Danvers, MA, USA). p53 (DO-1) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). V5 antibody was from Invitrogen (Grand Island, NY, USA). β-Actin antibody was from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase-conjugated goat anti-mouse secondary antibodies were obtained from Bio-Rad (Hercules, CA, USA). Goat anti-rabbit IgG (H + L) secondary antibody was from Invitrogen (Waltham, MA, USA). MG-132 was from Calbiochem (San Diego, CA, USA). Bortezomib was from Cayman Chemical (Ann Arbor, MI, USA). ONX 0914 was purchased from ApexBio (Houston, TX, USA). Actinomycin D and cycloheximide were from Sigma-Aldrich. All of the materials used in the experiments are in the highest grades and are commercially available.
7
RalR Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate RalR protein levels, Tricine-SDS-PAGE and a western blot were performed. BW25113 Δrac strains containing pCA24N, pCA24N-ralR, pCA24N-ralA and pCA24N-ralR-ralA were grown to a turbidity of 0.2 in LB with chloramphenicol, then 1 mM IPTG was added to produce RalR for 5 h, and cells were washed with TE buffer. Samples were sonicated and total protein was quantified as mentioned above. Protein was denatured at 95°C for 5 min. The Tricine-SDS-PAGE was performed as described earlier (43 (link)). Total protein (25 μg) of each sample was loaded for SDS-PAGE, and a western blot was performed using 2.5 μg protein of each sample with primary antibodies raised against a His-tag (Cell Signaling Technology, Danvers, MA, USA) and horseradish-peroxidase-conjugated goat anti-mouse secondary antibodies (Bio-Rad, Richmond, CA, USA).
8
Comprehensive Western Blotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed using the following antibodies: anti-Nog1 antibody (1:5000), anti-Nog2 antibody (1:20,000), anti-Arx1 antibody (1:2,000), anti-Nsa2 antibody (1:10,000), anti-Rlp24 antibody (1:2,000), provided by Micheline Fromont-Racine, anti-Nug1 antibody (1:10,000), anti-Bud20 antibody (1:5000), provided by Vikram Panse, anti-Ytm1 antibody (1:100), provided by John Woolford, anti-Has1 antibody (1:10,000) provided by Patrick Linder, anti-Ebp2 antibody (1:10,000), provided by Keiko Mizuta, anti-Noc3 antibody (1:1000), provided by Herbert Tschochner, anti-Rpl3 antibody (1:5,000), provided by Jonathan Warner, anti-Rsa4 antibody (1:10,000), provided by Miguel Remacha, anti-Arc1 antibody (1:5000, raised in the Hurt lab), horseradish-peroxidase-conjugated anti-Flag antibody (1:10,000; Sigma-Aldrich, Cat# A8592, RRID:AB_439702 ), horseradish-peroxidase-conjugated anti-HA antibody (1:5000; Roche, Cat# 12013819001; RRID:AB_390917 ), secondary horseradish-peroxidase-conjugated goat anti-rabbit antibody (1:2,000; Bio-Rad, Cat# 166-2408EDU, RRID:AB_11125345 ), and secondary horseradish-peroxidase-conjugated goat anti-mouse antibody (1:2,000; Bio-Rad, Cat# STAR105P, RRID:AB_323002 ).
9
Antibody Panel for Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed using the following antibodies: anti-Nog1 antibody (1:5000), anti-Nog2 antibody (1:20,000), anti-Arx1 antibody (1:2000), anti-Rei1 antibody (1:10,000), anti-Nsa2 antibody (1:10,000), anti-Rlp24 antibody (1:2000), provided by Micheline Fromont-Racine, anti-Nug1 antibody (1:10,000), anti-Yvh1 antibody (1:4000), provided by Vikram Panse, anti-Nmd3 antibody (1:10,000), anti-Rpl10 antibody (1:10,000), provided by Arlen Johnson, anti-Rpl3 antibody (1:5000), provided by Jonathan Warner, anti-Rpl5 antibody (1:10.000), provided by John Woolford, anti-Nop7 antibody (1:50,000), provided by Bruce Stillman, anti-Rsa4 antibody (1:10,000), provided by Miguel Remacha, anti-Mrt4 antibody (1:1000), provided by Juan Pedro Ballesta, anti-Arc1 antibody (1:5000), raised in our lab, anti-HA antibody (1:10,000, Covance Research Products, MMS-101R), horseradish-peroxidase-conjugated anti-Flag antibody (1:15,000, Sigma– Aldrich, A8592), secondary horseradish-peroxidase-conjugated goat anti-rabbit antibody (1:2000, Bio-Rad-170-6515), secondary horseradish-peroxidase-conjugated goat anti-mouse antibody (1:2000, Bio-Rad-170-6516).
10
Antibody-Based Protein Detection in Yeast
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed using the following antibodies: anti-Nog1 antibody (1:5000), anti-Nog2 antibody (1:5000), anti-Nsa2 antibody (1:5000), anti-Rlp24 antibody (1:2000), provided by Micheline Fromont-Racine, anti-Nug1 antibody (1:10 000), anti-Bud20 antibody (1:5000), provided by Vikram Panse, anti-Has1 antibody (1:10 000) provided by Patrick Linder, anti-Ebp2 antibody (1:10 000), provided by Keiko Mizuta, anti-Rpl3 antibody (1:5000), provided by Jonathan Warner, anti-Rsa4 antibody (1:10 000), provided by Miguel Remacha, anti-Mrt4 antibody (1:1000) provided by Juan Pedro Ballesta, horseradish-peroxidase-conjugated anti-Flag antibody (1:10 000; Sigma-Aldrich, Cat# A8592, RRID:AB_439702 ), horseradish-peroxidase-conjugated anti-HA antibody (1:5000; Roche, Cat# 12013819001; RRID:AB_390917 ), secondary horseradish-peroxidase-conjugated goat anti-rabbit antibody (1:2000; Bio-Rad, Cat# 166-2408EDU, RRID:AB_11125345 ), secondary horseradish-peroxidase-conjugated goat anti-mouse antibody (1:2000; Bio-Rad, Cat# STAR105P, RRID:AB_323002 ).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!