For western blot analysis of A6 cells transfected with siRNAs, cells were lysed in lysis buffer (50mM Tris-HCl pH7.4, 150 mM NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM DTT and protease inhibitor cocktail) 48hours after the second transfection. The lysates were sonicated on ice, centrifuged at 20,000 × g for 15 min, and then Laemmli buffer was added to the supernatant. The samples were heated at 98 °C for 5 min, and then subjected to SDS-PAGE with precast 4–15% Tris-HCl gradient SDS-PAGE gels (Bio-Rad). Proteins from the SDS-PAGE gels were then transferred onto polyvinylidene fluoride membranes (Bio-Rad). Western blotting was performed with a mouse monoclonal anti-Talin antibody (clone 8d4, Sigma-Aldrich, T3287) or a mouse monoclonal anti-ß-actin antibody (clone AC-74, Sigma-Aldrich, A2228) as the primary antibody and HRP-conjugated anti-mouse IgG antibody (eBioscience) as the secondary antibody.
Hrp conjugated anti mouse igg antibody
Manufactured by Thermo Fisher Scientific
Sourced in France
The HRP-conjugated anti-mouse IgG antibody is a secondary antibody that binds to the mouse primary antibody. It is conjugated with the enzyme horseradish peroxidase (HRP), which can be used for signal amplification in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Tris hcl gradient sds page gels, by Bio-Rad (2 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Anti talin antibody clone 8d4, by Merck Group (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Rsv g protein, by Sino Biological (1 mentions)
3 3 diaminobenzidine tetrahydrochloride substrate, by Thermo Fisher Scientific (1 mentions)
Rsv f protein, by Sino Biological (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Anti phospho p53 ser15, by Cell Signaling Technology (1 mentions)
Nitrocellulose blotting membrane, by GE Healthcare (1 mentions)
Anti actin, by Merck Group (1 mentions)
Hrp conjugated anti rabbit igg, by GE Healthcare (1 mentions)
Anti p16, by Santa Cruz Biotechnology (1 mentions)
Tmb substrate, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti rabbit igg antibody, by Merck Group (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Hrp conjugated anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
G box, by Syngene (1 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions)
17 protocols using hrp conjugated anti mouse igg antibody
1
Talin Expression Analysis in siRNA-Transfected A6 Cells
2
Western Blot Analysis of Talin in A6 Cells
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For western blot analysis of A6 cells transfected with siRNAs, cells were lysed in lysis buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM DTT and protease inhibitor cocktail) 48 h after the second transfection. The lysates were sonicated on ice, centrifuged at 20,000 × g for 15 min, and then Laemmli buffer was added to the supernatant. The samples were heated at 98 °C for 5 min, and then subjected to SDS-PAGE with precast 4-15% Tris-HCl gradient SDS-PAGE gels (Bio-Rad). Proteins from the SDS-PAGE gels were then transferred onto polyvinylidene fluoride membranes (Bio-Rad). Western blotting was performed with a mouse monoclonal anti-Talin antibody (clone 8d4, Sigma-Aldrich, T3287) at a dilution of 1:100 or a mouse monoclonal anti-ß-actin antibody (clone AC-74, Sigma-Aldrich, A2228) at a dilution of 1:1000 as the primary antibody and HRP-conjugated anti-mouse IgG antibody (eBioscience) at a dilution of 1:1000 as the secondary antibody.
3
Quantitative IFN-alpha Detection in Murine Serum
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For detection of IFNa in murine serum, we used 96-well flat-bottom microwell plates coated with monoclonal antibody to mouse IFNa (eBioscience) . The microwell strips were washed twice and then incubated with sample and biotin conjugate at room temperature for 2 h. Plates were washed with washing buffer and incubated with HRP-conjugated anti-mouse IgG antibody (eBioscience) for 60 min. Plates were washed and incubated with 1X tetramethylbenzidine (TMB) substrate solution (eBioscience) in the dark for 30 min, after which 10% H 2 SO 4 solution was added to stop the color reaction. Optical density was measured at 450 nm (FLUOstar Omega microplate reader, BMG LABTECH, Offenberg, Germany).
4
Antibody Response to VACV Infection
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BALB/c and C57BL/6 mice were intravenously injected via tail vein with single or multiple doses of virus at 5×106 pfu. In the multiple dosing groups, viruses were administered weekly three times. Blood was collected prior to the first dose of virus, then collected once weekly five times.
VACV-reactive antibodies were measured by ELISA method. Briefly, intact virus was coated on the plate and heat-inactivated virus-treated mouse serum was treated as primary antibody. Detection was performed using Horseradish Peroxidase (HRP)-conjugated anti-mouse IgG antibody (Invitrogen) (1:1000) at 450 nm to quantitate the total antibodies reactive to the coated virus. To measure the neutralizing activity by VACV-reactive antibodies, heat-inactivated mouse serum was incubated with virus, transferred onto cultures of U-2-OS cells, and incubated for 3 days. NAb titer50 was defined as the reciprocal of the highest dilution of serum that resulted in cell viability ≥50%.
VACV-reactive antibodies were measured by ELISA method. Briefly, intact virus was coated on the plate and heat-inactivated virus-treated mouse serum was treated as primary antibody. Detection was performed using Horseradish Peroxidase (HRP)-conjugated anti-mouse IgG antibody (Invitrogen) (1:1000) at 450 nm to quantitate the total antibodies reactive to the coated virus. To measure the neutralizing activity by VACV-reactive antibodies, heat-inactivated mouse serum was incubated with virus, transferred onto cultures of U-2-OS cells, and incubated for 3 days. NAb titer50 was defined as the reciprocal of the highest dilution of serum that resulted in cell viability ≥50%.
5
Quantification of RSV-Specific Antibodies and Viral Loads
6
Protein Expression in Irradiated Thyroid
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Thyroid samples from 4W and 7M rats at 1, 6, and 12 months after irradiation were analyzed for phospho-p53Ser15, p16, LC3, and p62 expression. Nonirradiated 4W and 7M thyroid tissues (controls) were removed at the same time and frozen immediately. Total protein was extracted from the tissues21 (link),35 (link). Proteins (30 µg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose blotting membranes (GE Healthcare, Tokyo, Japan). Membranes were incubated with anti-p16 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-p53Ser15 (Cell Signaling Technology, Danvers, MA, USA), anti-LC3, anti-p62/SQSTM1 (MBL), or anti-actin (Sigma-Aldrich, St. Louis, MO, USA) antibodies. This was followed by incubation with an HRP-conjugated anti-mouse IgG antibody (Invitrogen) or HRP-conjugated anti-rabbit IgG (GE Healthcare). Chemiluminescence (ECL Prime, GE Healthcare) was performed according to the manufacturer’s protocol. Protein detection was performed using LAS4000 (FUJIFILM) and quantified using NIH ImageJ software. Data are expressed as previously described21 (link).
7
Quantifying Antibody Responses to Plant-Expressed Proteins
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The purified plant-expressed BiP-MCP-Cor1-LysM-His protein was coated onto the surface of a 96-well microtiter plate at 4 °C overnight. After blocking at room temperature with 5% (w/v) skim milk in TBST buffer for 2 h, mice serum was diluted three-fold from 1:100 with 5% (w/v) skim milk in TBST buffer and incubated in a microplate for 2 h. After washing three times with TBST, horseradish peroxidase (HRP)-conjugated anti mouse IgG antibody (Invitrogen, Cat# 32,430, 1:5,000 dilution) was added and incubated for 2 h. After three washes with TBST, TMB substrate (Thermo Fisher, Cat# 34,025) was added followed by 0.1 M H2SO4 to stop the reaction. The signal intensity was measured at an absorbance of 450 nm using multi-microplate readers (Perkin Elmer).
8
Western Blot Analysis of Inflammasome Components
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Cell lysates were separated by 10% or 12% SDS-PAGE, transferred to PVDF membranes, and probed with anti-NLRP3 antibody (CST, Cat#15101S), anti-β-actin antibody (CST, Cat#4970S), anti-GSDMD antibody (CST, Cat#39754S) and anti-caspase-1 antibody (AdipoGen, Cat#AG-20B-0042-C100). HRP-conjugated anti-Rabbit IgG antibody (Sigma, Cat#A0545) or HRP-conjugated anti-mouse IgG antibody (Invitrogen, Cat#31,430) was used as a secondary antibody. Immunoblots were developed using a chemiluminescent HRP-conjugated substrate (Millipore, Cat#WBKLS500) and imaged on a ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories).
9
Western Blot Analysis of Leishmania Proteins
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Leishmania cell lysates were prepared by re-suspending and boiling the cell pellets for 5 minutes in an SDS-PAGE sample buffer. Samples were resolved on SDS-polyacrylamide gel by electrophoresis and electro-blotted onto nitrocellulose membrane (0.45μm, Synergy scientific services) in Tris-glycine buffer (pH 8.3) at 80V for 3 hours. The membrane was treated with 5% skimmed milk to block the nonspecific sites, and then probed with rabbit anti-LdPfn antibodies [11 (link)] (1:100 dilution) or mouse anti-β tubulin monoclonal antibodies (Sigma, cat.No. T7816) (1:5000) or rabbit anti-eIF4A.1 antibodies (Cell Signalling Technology, cat No. 2490) (1:100) for overnight at 4°C. The membrane was washed 5 times with Tris-buffered saline (pH 7.5) containing 0.05% (v/v) Tween 20, then incubated with HRP conjugated anti-rabbit IgG antibody (Invitrogen, cat no. A16074) or HRP conjugated anti-mouse IgG antibody (Invitrogen, cat no.62-6820) for 2 hours and developed with ECL (Bio-Rad, Clarity) and imaging software (Syngene, G-box).
10
Proteinase-K Digestion of Brain Homogenates
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10% brain homogenates were prepared in 0.32 M sucrose by using a Precellys24 (Bertin). Extracts of 50–90 μg protein were digested with 50 μg/ml proteinase-K in DOC/NP-40 0.5% for 45 minutes at 37°C. The reaction was stopped by adding 3 μl complete protease inhibitor cocktail (7x concentrated) and 8 μl of a lauryl dodecyl sulfate (LDS)-based sample buffer. The samples were heated to 95°C for 5 minutes prior to electrophoresis through a 12% Bis-Tris precast gel (Invitrogen), followed by transfer to a nitrocellulose membrane by wet blotting. Proteins were detected with anti-PrP POM-1 antibody (1:10000). For secondary detection, an HRP-conjugated anti-mouse IgG antibody (Zymed, Invitrogen) was used and signals were visualized with an ECL detection kit (Pierce).
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