A Salmonella strain expressing 3 × FLAG-tagged NagB was cultured under either the Pi+ or Pi− media as described above. Gel-separated bacterial proteins were transferred to the PVDF membrane and probed with primary antibodies specific for FLAG (Sigma, St. Louis, MO, USA) (1:5000) and anti-mouse HRP-conjugated secondary antibodies (Sigma, St. Louis, MO, USA) (1:5000). As a loading control, DnaK was probed by using Salmonella anti-DnaK (Enzo Life Sciences, New York, NY, USA) (1:5000) and anti-mouse HRP-conjugated secondary antibodies (Sigma, St. Louis, MO, USA) (1:5000).
Anti mouse hrp conjugated secondary antibody
Manufactured by Merck Group
Sourced in United States, United Kingdom
The Anti-mouse HRP-conjugated secondary antibody is a laboratory reagent used in various immunoassay techniques. It consists of an antibody against mouse immunoglobulins that is conjugated with the enzyme horseradish peroxidase (HRP). This reagent can be used to detect and quantify the presence of mouse-derived primary antibodies in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Ecl system, by GE Healthcare (7 mentions)
Hybond, by Cytiva (5 mentions)
Immobilon p transfer membrane, by Merck Group (3 mentions)
Anti rabbit hrp conjugated secondary antibody, by Merck Group (3 mentions)
Anti flag m2, by Merck Group (2 mentions)
Anti actin hrp coupled antibodies, by Merck Group (2 mentions)
Anti gfp antibody, by Roche (2 mentions)
Mouse monoclonal anti ha antibody 12ca5, by Roche (2 mentions)
Mouse monoclonal anti flag antibody, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Amersham ecl detection reagent, by GE Healthcare (2 mentions)
Mouse anti tubulin primary antibody, by Merck Group (2 mentions)
Amersham ecl, by Cytiva (2 mentions)
Hybond xl, by Cytiva (2 mentions)
Luminata hrp substrate, by Merck Group (2 mentions)
Anti β actin, by Merck Group (1 mentions)
Complete mini, by Roche (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ecl plus detection system, by GE Healthcare (1 mentions)
37 protocols using anti mouse hrp conjugated secondary antibody
1
Detecting Salmonella NagB Expression
2
Immunoblotting Analysis of COS-1 Cells
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COS-1 cells were harvested 24–48 hours after transfection and resuspended in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA and 1% NP40) supplemented with Complete Mini (Roche, Switzerland) for protein extraction. Protein concentrations were determined using the BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein extracts (200 μg) were fractionated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, transferred to an Immobilon-P membrane (Millipore, Billerica, MA, USA) and incubated with primary specific antibodies anti-Flag M2 and anti-β-actin (Sigma-Aldrich Co., St Louis, MO, USA). The ECL Plus detection System (GE Healthcare, Buckinghamshire, UK) with HRP-conjugated anti-mouse secondary antibodies (Sigma-Aldrich Co.) was used for detection.
3
Western Blot Analysis of CbAgo
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The level of CbAgo expression and the amount of CbAgo used for smDNA purification was determined by Western blotting. Protein samples were mixed with 2x Laemmli sample buffer (120 mM Tris-HCl, 4% SDS, 4% b-mercaptoethanol, 10% Glycerol, pH 6.8) and heated at 95°C for 5 min, and then resolved by electrophoresis in a 4-20% Tris-glycine gel (BioRad).
Proteins were transferred onto a nitrocellulose membrane in Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) using semi-dry procedure at 25 V, 1 A for 30 min (BioRad Trans-Blot Turbo). The transfer membrane was air-dried and then washed in PBS (10 mM phosphate buffer, 137 mM NaCl, 2.7 mM KCl) for 5 min. The membrane was blocked with blocking buffer (PBS, Tween-20 0.1% (v/v), non-fat milk 5% (m/v)) for 30 min at room temperature, and then incubated with anti-6xHis monoclonal antibodies (1:1000, Sigma) for 1 h at room temperature. The membrane was washed 4 times with PBST buffer (PBS, Tween-20 0.1% (v/v)), and after that incubated with HRP-conjugated anti-mouse secondary antibodies (1:10000, Sigma) for 1 h at room temperature and washed again as described above. Antigen-antibody complexes were detected with Immobilon ECL Ultra Western HRP substrate (Millipore) on a Chemidoc XRS+ imager (BioRad).
Proteins were transferred onto a nitrocellulose membrane in Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) using semi-dry procedure at 25 V, 1 A for 30 min (BioRad Trans-Blot Turbo). The transfer membrane was air-dried and then washed in PBS (10 mM phosphate buffer, 137 mM NaCl, 2.7 mM KCl) for 5 min. The membrane was blocked with blocking buffer (PBS, Tween-20 0.1% (v/v), non-fat milk 5% (m/v)) for 30 min at room temperature, and then incubated with anti-6xHis monoclonal antibodies (1:1000, Sigma) for 1 h at room temperature. The membrane was washed 4 times with PBST buffer (PBS, Tween-20 0.1% (v/v)), and after that incubated with HRP-conjugated anti-mouse secondary antibodies (1:10000, Sigma) for 1 h at room temperature and washed again as described above. Antigen-antibody complexes were detected with Immobilon ECL Ultra Western HRP substrate (Millipore) on a Chemidoc XRS+ imager (BioRad).
4
Western Blot Analysis of Tagged Proteins
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Separated proteins on PAGE gels were transferred onto PVDF membranes (0.2 μm) using a Trans-Blot Turbo RTA Transfer Kit (Bio-Rad). Blots were washed for 1 min with TBST followed by blocking with Superblock (Thermo-Fisher) for 30 min. Afterwards, blots were incubated overnight at 4 °C with primary antibodies anti-Strep-tag II (1:2000; NBP2-43735 Novus biologicals) or anti-FLAG (1:2000; F1804 Sigma). Unbound antibodies were removed via three 5 min washes with TBST followed by incubation of the blot for 1 h with the HRP-conjugated anti-mouse secondary antibodies (1:10,000; A9044, Sigma-Aldrich). After three successive washes with TBST and one wash with TBS, Thermo Scientific™ SuperSignal™ West Pico PLUS Chemiluminescent Substrate ECL substrate (Thermo) was added, and signal detection was obtained with an ImageQuant™ LAS 4000 (Amersham).
5
Striatal Protein Analysis by SDS-PAGE and WB
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SDS-PAGE and western blotting experiments were performed as previously described [40 (link)]. Striatal tissues were isolated and homogenized in lysis buffer (150 mM NaCl, 50 mM Tris, 1% Triton) supplemented with protease and phosphatase inhibitors. Proteins were separated using the NuPAGE system on 4–12% pre-cast gradient gels and MES/SDS running buffer (Thermo Fisher Scientific, Waltham, MA) and then were transferred to Immobilon P transfer membrane (Millipore, Burlington, MA) using a Mini Blot Module (Thermo Fisher Scientific, Waltham, MA). Blots were probed with anti-tyrosine hydroxylase (TH) antibodies (MAB318, Millipore, Burlington, MA) and anti-actin HRP-coupled antibodies (A3854, Sigma-Aldrich, St. Louis, MO). HRP-conjugated anti-mouse secondary antibodies (Millipore, Burlington, MA) were used to detect TH immunoreactivity in association with chemiluminescence-based detection, ECL-Plus (Thermo Fisher Scientific, Waltham, MA).
6
Striatal Protein Analysis by SDS-PAGE and Western Blotting
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SDS-PAGE and Western blotting experiments were performed as previously described [40] . Striatal tissues were isolated and homogenized in Lysis buffer (150mM NaCl, 50mM Tris, 1% Triton) supplemented with protease and phosphatase inhibitors. Proteins were separated using the NuPAGE system on 4-12% precast gradient gels and MES/SDS running buffer (Thermo Fisher Scienti c, Waltham, MA) and then were transferred to Immobilon P transfer membrane (Millipore, Burlington, MA) using a Mini Blot Module (Thermo Fisher Scienti c, Waltham, MA). Blots were probed with anti-tyrosine hydroxylase (TH) antibodies (MAB318, Millipore, Burlington, MA) and anti-actin HRP-coupled antibodies (A3854, Sigma-Aldrich, St. Louis, MO). HRP-conjugated anti-mouse secondary antibodies (Millipore, Burlington, MA) were used to detect TH immunoreactivity in association with chemiluminescence-based detection, ECL-Plus (Thermo Fisher Scienti c, Waltham, MA).
7
Quantification of Anti-Rabies Antibody
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The levels of antibody in the experimental mice were quantified by ELISA at 7, 14, and 21 dpi. Sera were added to the previously antigen-coated plate and incubated at 37°C for 1 h. Following the incubation period, plates were washed 3 times with PBST and incubated with HRP conjugated anti-mouse secondary antibody 1:3000 (V/V) (Sigma, USA) at 37°C for 1 h. Following the incubation period, the plates were washed as described above and finally developed with the substrate solution (100 mM citrate-phosphate buffer containing 1 mg/ml O-phenylenediamine and 1 µl/ml of 30% of H2O2). Finally, after 15 min, the reaction was stopped by adding 50 µl of 8N H2SO4, and the absorbance was measured at 490 nm in ELISA reader (Bio-Rad, USA) [16 (link)].
The level of anti-rabies antibody was expressed as mean absorbance at optical density 490 nm (OD 490 nm). To determine the cutoff, the OD values of the sera from the control mice were taken, and the mean OD value was calculated. The mean OD value + 3 standard deviation (SD) was considered as the cutoff value, above which the sera samples were considered as positive.
The level of anti-rabies antibody was expressed as mean absorbance at optical density 490 nm (OD 490 nm). To determine the cutoff, the OD values of the sera from the control mice were taken, and the mean OD value was calculated. The mean OD value + 3 standard deviation (SD) was considered as the cutoff value, above which the sera samples were considered as positive.
8
Western Blot Analysis of Cell Lysates
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Cells were lysed in 1 × SDS-PAGE sample buffer (0.2 M Tris–HCl, pH 6.8, 8% SDS, 0.1% bromophenol blue, 40% glycerol, 20% β-mercaptoethanol; all chemicals are from Sigma-Aldrich). The protein lysates were centrifuged at 4°C for 10 min at 10,000 × g and then boiled at 95°C for 10 min. Protein extracts were separated on 10% SDS-PAGE gel and transferred to a PVDF membrane (BioRad Laboratories). Blots were blocked in TBST with 5% BSA for 1 h, followed by overnight incubation with a primary antibody at 4°C. The primary antibodies used were NQO1 (#11451-1-AP; Proteintech), WWP2 (#15469-1-AP; Proteintech), EDN1 (#ab2786; Abcam), and β-actin (#3700S; Cell signaling). Blots were further incubated with HRP-conjugated anti-mouse secondary antibody (#401253; Sigma-Aldrich) at room temperature for 1 h. Protein bands were developed with the ECL system (Pierce) and performed by ChemiDoc MP Imager (Bio-rad Laboratories). The protein band density was analyzed using ImageJ.
9
Visualizing dsRNA and p14 Proteins
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Diluted dsRNA (E200 synthesized as described previously [21 (link)]) or purified p14 were applied to a dry Hybond-N+ positively charged nylon membrane (Amersham) and crosslinked with 100 µJ/cm2 UV light using a Stratalinker 1800 (Stratagene). The membrane was blocked in 5% skim milk in TBS for 1 h, incubated with J2 antibody (SCICONS) diluted 1:2000 in 1% BSA in TBS for 2 h, followed by HRP-conjugated anti-mouse secondary antibody (Sigma, St. Louis, MO, USA) diluted 1:5000 in 1% skim milk in TBS for 1 h. An ECL solution of luminol, 4-IPBA and H2O2 in 100 mM Tris, as described [22 (link)], was added prior to X-ray film exposures.
10
Quantifying Hybridoma Binding to MHC-I
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Ninety-six well MaxiSorp plates (Sigma-Aldrich 44-2404-21) were coated with 100μl of streptavidin at 10μg/ml in PBS at 4°C overnight. The plates were then washed with PBS/0.1% Tween-20 and blocked with 1% BSA in PBS for 2h at room temperature. Plates were used fresh or kept at -20°C after washing for future use.
To test hybridoma binding to MHC-I/peptide complexes, plates were used fresh or recovered from the -20°C freezer and thawed at room temperature. MHC-I/peptide monomers were added to the wells at 1μg/ml (100μl) and incubated for 1h at room temperature. After washing with PBS-Tween 20, 100μl of mAb at 10μg/ml or neat hybridoma supernatants were added to the wells and incubated for 1h. After washing with PBS-Tween 20, HRP conjugated anti-mouse secondary antibody (Sigma-Aldrich A9044) was added at 1:1000 dilution to each well and incubated for 1h. Substrate ABTS Solution (Roche 10102946001) was added to each well (100μl) after washing and OD405nm was measured with a plate reader within 5-30min.
To test hybridoma binding to MHC-I/peptide complexes, plates were used fresh or recovered from the -20°C freezer and thawed at room temperature. MHC-I/peptide monomers were added to the wells at 1μg/ml (100μl) and incubated for 1h at room temperature. After washing with PBS-Tween 20, 100μl of mAb at 10μg/ml or neat hybridoma supernatants were added to the wells and incubated for 1h. After washing with PBS-Tween 20, HRP conjugated anti-mouse secondary antibody (Sigma-Aldrich A9044) was added at 1:1000 dilution to each well and incubated for 1h. Substrate ABTS Solution (Roche 10102946001) was added to each well (100μl) after washing and OD405nm was measured with a plate reader within 5-30min.
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