Worms were collected at the first or fifth day of adulthood, in M9 buffer (45 mM KH2PO4, 42 mM Na2HPO4, 85 mM NaCl, 1 mM MgSO4 in water) and lysed by sonication in lysis buffer (25 mM Tris-HCl pH 7.5, 5 mM NaCl, 5 mM EDTA, 1 mM DTT, protease inhibitor cocktail Roche Applied Science). For each lysate, equal amounts of total proteins, quantified with the Pierce BCA Protein Assay Kit (ThermoScientific), were loaded onto either a 4–20% Mini-PROTEAN TGX (Biorad) or 8–18% Excel SDS gel (GE Healthcare) for electrophoresis performed under reducing conditions. Proteins were transferred to Immobilon P membranes (Millipore) and blocked with 5% non-fat milk, in tris-buffered saline and Tween 20 (TBS-T), for one hour. Western blots were developed with 4.6 μg/ml rabbit polyclonal anti-human β2-m antibody (A0072, Dako) O.N. at 4 °C and 1.3 ng/ml anti-rabbit IgG peroxidase conjugate (A0545 Sigma) for 1 h RT, as primary and secondary antibody respectively. To normalize the content of total protein, western blot was developed with 0.185 μg/ml anti-glyceraldehyde 3-phosphate dehydrogenase antibody (anti-GAPDH selected as loading control, ab181602 Abcam) O.N. at 4 °C, and 1.3 ng/ml secondary anti-rabbit IgG peroxidase conjugate (A0545 Sigma) antibody for 1 h RT. Immunoreactive bands were detected by ECL chemio-luminescence (Millipore), and quantified with Image Studio Lite (LI-COR Biosciences).
Anti rabbit igg peroxidase conjugate
Manufactured by Merck Group
Sourced in United States
Anti-rabbit IgG peroxidase conjugate is a laboratory reagent used as a detection tool in immunoassays. It is composed of antibodies specific to rabbit immunoglobulin G (IgG) that are conjugated to the enzyme peroxidase. This conjugate can be used to identify the presence of rabbit IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igg peroxidase conjugate, by Merck Group (5 mentions)
Anti β actin, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Image studio lite, by LI COR (1 mentions)
Anti human β2 m antibody, by Agilent Technologies (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Mini protean tgx, by Bio-Rad (1 mentions)
Myriocin, by Merck Group (1 mentions)
Calcoflour white cfw, by Merck Group (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Quinacrine, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Analytical grade solvents, by Merck Group (1 mentions)
Restriction enzymes, by Promega (1 mentions)
Cell culture medium, by Thermo Fisher Scientific (1 mentions)
Pcdna3 plasmid, by Thermo Fisher Scientific (1 mentions)
Silica gel 60 chromatography plates, by Merck Group (1 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions)
17 protocols using anti rabbit igg peroxidase conjugate
1
Quantification of β2-Microglobulin in C. elegans
2
Lipid Trafficking and Stress Response
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Aureobasidin A was obtained from Takara Shuzo Co, tunicamycin from Sigma Aldrich, FM4-64 from Molecular probes (T-13320), dihydroethidium (DHE) from Marker gene technologies. Calcoflour white (CFW), myriocin, quinacrine, and N-acetyl-L-cysteine (NAC) from Sigma-Aldrich, [3H]myo-inositol from ANAWA Trading SA. Anti-Kar2 and anti-Gas1 antibodies were the kind gifts of Drs. M. Rose and F.Reggiori, anti-CPY antibodies (A-6428) were from Molecular Probes. Secondary antibodies were anti-rabbit IgG peroxidase conjugate (Sigma A6154) and anti-mouse IgG peroxidase conjugate (Sigma A4416). PVDF membranes were obtained from Millipore, Cat.No IPVH00010.
3
Cell Culture and Molecular Techniques
4
Quantitative Immunoblotting Analysis of Muscle Proteins
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Equal amounts of muscle lysates (50 μg of protein per sample) were separated on 4–20% SDS-polyacrylamide gradient gels and transferred onto a polyvinylidene difluoride membrane. The analysis procedure was performed according to [58 (link)]. The following antibodies were used: rabbit monoclonal IgG anti-SDH (cat. No. #11998, 1:1000; Cell Signaling, Beverly, MA, USA); rabbit polyclonal anti-PGC-1α (cat. no. sc-13067, 1:500; Santa Cruz Biotechnology, Dallas, TX, USA); anti-rabbit IgG–peroxidase conjugate (cat. no. A9169, 1:25,000; Sigma Aldrich, St. Louis, MI, USA). After incubation in primary (overnight at 4°C) and secondary antibodies (1 h at room temperature), immunoblots were detected and visualized using enhanced chemiluminescence reagents (Western Lightning Plus ECL, Perkin Elmer, Waltham, MA, USA). Changes in protein levels were assessed by densitometry of the immunoreactive bands and normalized to the total amount of protein in the samples transferred onto the membrane (Figure S1 ) [59 (link)]. Relative protein levels were analyzed and quantified using ChemiDoc image analysis system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The immunoblotting analyses were done for six randomly selected animals from each group.
5
Extraction and Detection of T1P
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The bacterial suspensions were adjusted to an absorbance of 0.7 at the optical density (OD) at 600 nm (OD600). Equal numbers of bacteria were used to prepare whole cell extracts. To dissociate T1P from the bacteria, the cultures were treated with acidified water (pH 1.8), boiled for 10 min in denaturation sample buffer, neutralized to pH 7.2, and then resolved by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with rabbit anti-T1P (1:3,000) in PBS-Tween 80 for 1 h followed by anti-rabbit IgG-peroxidase conjugate (Sigma; 1:5,000) and the reaction was visualized by addition of a chemiluminescent substrate (Amersham). Anti-DnaK antibody was used to control for the amount of protein loaded in the gels.
6
Chromatin Immunoprecipitation (ChIP) Assay
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ChIP experiments were carried out as previously described (16 (link)). Cells were treated with 1% formaldehyde for 15 min at 37ºC for crosslinking. Immunoprecipitations (IPs) were performed using the following reagents: Total RNAPII: Dynabeads Protein A (Invitrogen) and rabbit polyclonal anti-RPB1 N-20 (Santa Cruz); RPB1 CTD-Ser5P: Dynabeads Protein G (Invitrogen) and anti-RNA polymerase II (phospho-CTD-Ser5) antibody clone 3E8 (Millipore); NELF-E: Dynabeads Protein A (Invitrogen) and rabbit polyclonal anti-NELF-E (Santa Cruz). Primers used are listed in Supplementary Table S1.
Antibodies used for Western blotting are rabbit polyclonal anti-NELF-E (sc-32912, Santa Cruz Biotech.), rabbit polyclonal anti-SNF2H (A301–017A, Bethyl), mouse monoclonal anti-α-Tubulin and, as secondary antibodies, anti-Rabbit IgG peroxidase conjugate (Sigma) and anti-Mouse IgG peroxidase conjugate (Sigma).
Antibodies used for Western blotting are rabbit polyclonal anti-NELF-E (sc-32912, Santa Cruz Biotech.), rabbit polyclonal anti-SNF2H (A301–017A, Bethyl), mouse monoclonal anti-α-Tubulin and, as secondary antibodies, anti-Rabbit IgG peroxidase conjugate (Sigma) and anti-Mouse IgG peroxidase conjugate (Sigma).
7
Western Blot Protein Analysis Protocol
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The separation of proteins was performed, accordingly to their size, in denaturing conditions, through SDS-PAGE vertical electrophoresis. Proteins were transferred to PVDF Immobilon-P or Immobilon-FL membranes (Millipore) as previously reported [28 (link), 36 , 47 (link), 67 (link)]. PDVF membranes were incubated with the specific primary antibody (Table 1 ). The secondary antibodies Goat Anti-Mouse IgG, DyLight 680 (red colored) and Goat Anti-Rabbit IgG, DyLight 800 (green colored) (Thermo Scientific) were incubated at 1:10,000 dilutions in TBS-T, during 1 h (in the dark). Next, membranes were washed with TBS-T (3×, 10 min washes) and scanned in the LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences; Lincoln, NE, USA) that detects the fluorescence associated with the secondary antibody. The secondary antibodies were either Anti-Mouse IgG-Horseradish Peroxidase-Linked Species-Specific Whole Antibody (Amersham Biosciences; Amersham, UK) or Anti-Rabbit IgG-Peroxidase Conjugate (Sigma-Aldrich). In these cases, after a 1 h incubation with the secondary antibody (1:10,000), the luminescence was detected with the ECL Western Blotting Detection Reagent.
8
Western Blot Analysis of Luciferase in Cox2 Mice
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For western blot analysis, gastrocnemius muscles from Cox2FLuc/+ mice were homogenized in reducing sample buffer (80 mM Tris, pH 6.8, 0.1 M dithiothreitol, 2% SDS, and 10% glycerol with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA)) using a Dounce homogenizer. Protein extracts were boiled for 1 minute, placed on ice, and loaded to SDS PAGE (40 μg per well) followed by transfer to nitrocellulose membrane (100 V, 1 h, 4°C). As a positive control, 7.5 ng of QuantiLum recombinant luciferase (Promega, E1701) was used. Membranes were stained with Ponceau red and imaged and then blocked with 3% BSA and probed with anti-luciferase antibody (Promega, 1:1,000) followed by anti-rabbit IgG peroxidase conjugate (Sigma-Aldrich). Blots were developed using ChemiGlow West substrate (ProteinSimple, San Jose, CA, USA) and detected using an AlphaImager gel documentation system (formerly Alpha Innotech, now ProteinSimple). The actin band from the Ponceau stained membrane served as the loading control.
9
Detecting SARS-CoV-2 Spike Protein in HEK Cells
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Approximately 105 number of HEK cells were seeded per well in a 12-well plate and incubated for 16 h at 37°C with 5% CO2. Post-incubation the HEK cells were incubated with 100 µL of 10-1 and 10-2 dilutions of BBV154 vector for 45 min at 37°C with 5% CO2. Inoculum was removed and 1 mL of MEM with 1% FBS was added to each well and incubated at 37°C with 5% CO2 for 48 h. The spent medium was removed from the cells and ice-cold methanol was added to the cells and incubated at -20°C for 20 min. The fixed cells were washed with PBS and incubated with rabbit polyclonal S1 antibody. Anti-rabbit IgG peroxidase conjugate was used as the secondary antibody (Sigma-Aldrich, USA). Insoluble chromogen; 3-amino-9-ethylcarbazole (AEC) was used for the detection of spike in the BBV154 infected HEK cells. Cells expressing spike are expected to be stained with AEC as dark pink cells.
10
Proteomic Analysis of Protein Complexes
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