To assess the activity of the ITIM signaling pathway, 2–5x106 cells isolated splenic B cells or BMDMs were washed in RPMI and cultured in supplemented RPMI before addition of irrelevant, hFcγRIIB agonist (6G08) or hFcγRIIB antagonist (6G-Q) mAb (10 μg/mL) in hIgG1 N297Q formats at 37°C for either 30 min (B cells) or 15 min (BMDMs). Cells were subsequently washed with cold PBS and lysed in Onyx buffer (containing a cocktail of protease and phosphatase inhibitors). Samples were separated by SDS PAGE on 8–10% Bis-Tris gels (Invitrogen) in MOPS running buffer. Gels were transferred to a PDVF membrane using the iBlot 2 Transfer System (Invitrogen) and blocked in 5% BSA-TBS-T for 1 h. The membrane was then incubated with primary antibodies for 16 h at 4°C, washed and then incubated with HRP-conjugated secondary IgG (Sigma Aldrich) for 1.5 h at RT. ECL substrate (GE Healthcare) was used for detection and samples were imaged using the Chemi Doc-it imaging system (UVP). Antibodies against hFcγRIIB (EP888Y, Abcam) phosphorylated hFcγRIIB (EP926Y, Abcam), SHIP-1 (2728, Cell Signaling Technology), phosphorylated SHIP1 (3941, Cell Signaling Technology) and alpha-tubulin (2144, Cell Signaling Technology) were used for Western Blotting.
Hrp conjugated secondary igg
Manufactured by Merck Group
Sourced in United States
HRP-conjugated secondary IgG is a laboratory reagent used in immunoassays and western blotting. It consists of a horseradish peroxidase (HRP) enzyme conjugated to a secondary antibody that binds to the primary antibody used in the experiment. This allows for the detection and visualization of target proteins or antigens through an enzymatic reaction.
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Lab products found in correlation
Ecl detection system, by Thermo Fisher Scientific (6 mentions)
Quantity one software, by Bio-Rad (5 mentions)
Chemidoc, by Bio-Rad (4 mentions)
Sds page, by Thermo Fisher Scientific (2 mentions)
β actin, by Fortis Life Sciences (2 mentions)
Iblot2 transfer system, by Thermo Fisher Scientific (1 mentions)
Ep888y, by Abcam (1 mentions)
Ecl substrate, by GE Healthcare (1 mentions)
Hif 1α, by Fortis Life Sciences (1 mentions)
Phosphor p70s6k, by Cell Signaling Technology (1 mentions)
Phospho ampk, by Cell Signaling Technology (1 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Anti aif, by Cell Signaling Technology (1 mentions)
Anti caspase 9, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Anti nrf2, by Cell Signaling Technology (1 mentions)
9 protocols using hrp conjugated secondary igg
1
ITIM Signaling Pathway Evaluation
2
Western Blot Analysis of Autophagy Markers
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Cells or isolated mitochondria were lysed in the RIPA buffer containing Na3VO4 (1 mM) and protease inhibitors (1 μg/ml). The cell lysate proteins were separated using SDS-PAGE and transferred onto nitrocellulose membranes (Protran). After blocking with 5% non-fat milk in PBS-Tween (0.1%), the membranes were incubated with antibodies against IRS1, HIF1α (both Bethyl), BECN1, SQSTM1/p62, LC3B, phosphor-p70S6K, phospho-AMPK, AMPK, phospho-ULK1 (Ser-555), phospho-ULK1 (Ser-757), and ULK1 (all Cell Signaling), phospho-mTOR (p-mTOR, Ser-2448), mTOR, PARK2 (all ThermoFisher Scientific), and GLUT-4, cytochrome c and VDAC1 (all Santa Cruz). After incubation with the HRP-conjugated secondary IgG (Sigma), blots were developed using the ECL detection system (Pierce Biotechnology). The band intensities were visualized and quantified with ChemiDoc using the Quantity One software (BioRad Laboratories).
3
H7N9 Influenza HA-specific IgG Titers
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HA-specific IgG antibody titers in the sera collected at Week 3 p.v. were determined by enzyme-linked immunosorbent assay (ELISA) as described previously (51 (link)). In brief, the flat-bottomed 96-well microplate plates were coated with 250 ng of the purified HA protein of H7N9 A/Anhui/1/2013 (AH13) virus (Sino Biological) or the purified TT3 HA protein expressed in Sf9 insect cells at 4°C overnight. PBST (PBS containing 0.05% Tween 20) supplemented with 1% bovine serum albumin (BSA) were added for blocking at 37°C for 2 h. The chicken sera were serially diluted, and then added to the well and incubated at 37°C for 1 h. Then, the plates were washed with PBST three times and incubated with 100 μL of HRP-conjugated secondary IgG (Sigma, St. Louis, USA) at 37°C for 1 h. The plates were then washed with PBST three times and incubated with 100 μL of 3, 3', 5, 5'-tetramethylbenzidine (TMB) liquid substrate. The reaction was stopped by the addition 50 μL of 2M H2SO4 and the optical density (OD) was read at 450 nm using a spectrophotometer.
4
Immunoblotting of Exosomal CD81
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For immunoblotting, exosomes (10 µg protein) were resolved using 4–12% SDS-PAGE (Life Technologies), transferred to nitrocellulose membranes, and incubated overnight with anti-CD81 (generously provided by Prof. Fabio Malavasi, University of Torino, Torino, Italy). Actin (Bethyl) was used as loading control. After incubation with an HRP-conjugated secondary IgG (Sigma), the blots were developed using the ECL detection system (Pierce Biotechnology). Intensities of the individual bands were visualized by ChemiDoc using the Quantity One software (BioRad).
5
Western Blot Analysis of Nuclear Translocation
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For western blot analysis, protein samples (50 μg per lane) were resolved using 12.5% SDS-PAGE, and transferred to nitrocellulose membranes, and incubated overnight with anti-p65 (Cell Signaling Technology, Danvers, MA). β-Actin and lamin (Bethyl, Montgomery, TX, USA) were used as standing loading controls in the Western Blotting analysis of both cytosolic and nuclear fractions respectively. HRP-conjugated secondary IgG was incubated (Sigma) and the blots were developed using the ECL detection system (Pierce Biotechnology, Rockford, IL, USA). Quantity One software was used to quantify band intensities were visualized by ChemiDoc system (BioRad, Hercules, CA).
6
IRS1, MAPK, and ERK Signaling Immunoblotting
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Cells were lysed in the RIPA buffer containing Na3VO4 (1 mM) and protease inhibitors (1 μg/ml). The cell lysate proteins were separated using SDS-PAGE and transferred onto nitrocellulose membranes (Protran). After blocking with 5% non-fat milk in PBS-Tween (0.1%), the membranes were incubated with antibodies against IRS1 (Bethyl), phospho-p38 MAPK, p38-MAPK, phospho-ERK1/2 and ERK1/2 (all Cell Signaling). β-actin was used as a loading control. After incubation with the HRP-conjugated secondary IgG (Sigma), blots were developed using the ECL detection system (Pierce Biotechnology). The band intensities were visualized and quantified with ChemiDoc using the Quantity One software (BioRad Laboratories).
7
Apoptotic Pathway Analysis in Cancer Cells
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MDA-MB-231 and PC3 cells (3×105 per well in 6-well plates) were treated with α-TOS (60μM) and AMP compounds (30 μM) for 180 min, harvested, and the pellet re-suspended in the digitonin cell permebilization buffer (Trevigen, Gaithersburg, MD). The supernatant containing the cytosolic fraction was collected. The remaining pellet (organelle fraction) was lysed in the RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4,1 μg/ml protease inhibitors).
For western blot analysis, protein samples (50 μg per lane) were resolved using 12.5% SDS-PAGE, and transferred to nitrocellulose membranes, and incubated overnight with anti-caspase-9, anti-caspase-3 and anti-AIF (Cell Signaling Technology). β-Actin (Bethyl, Montgomery, TX, USA) was used as a loading control. After incubation with an HRP-conjugated secondary IgG (Sigma), the blots were developed using the ECL detection system (Pierce Biotechnology, Rockford, IL, USA). Band intensities were visualized by ChemiDoc using the Quantity One software (BioRad).
For western blot analysis, protein samples (50 μg per lane) were resolved using 12.5% SDS-PAGE, and transferred to nitrocellulose membranes, and incubated overnight with anti-caspase-9, anti-caspase-3 and anti-AIF (Cell Signaling Technology). β-Actin (Bethyl, Montgomery, TX, USA) was used as a loading control. After incubation with an HRP-conjugated secondary IgG (Sigma), the blots were developed using the ECL detection system (Pierce Biotechnology, Rockford, IL, USA). Band intensities were visualized by ChemiDoc using the Quantity One software (BioRad).
8
Nuclear Fractionation and Western Blot
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Cells (3×10 5 per well in 6-well plates) were harvested, and the pellet re-suspended in the cytoplasm extract (CE) buffer (10 mM HEPES, 60 mM KCl, 1 mM EDTA, 0.075% (v/v) NP40, 1mM DTT and 1 mM PMSF, adjusted to pH 7.6). After 5 min incubation on ice, the pellet was centrifuged at 1500 rpms for 5 min, and the supernatant containing the cytosolic fraction obtained. The remaining pellet was washed in CE buffer, and lysed in the nuclear extract (NE) buffer (20 mM Tris-HCl, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM PMSF and 25% (v/v) glycerol, adjusted to pH 8.0). After 20 min incubation on ice, the pellet was centrifuged at 12000 rpms for 10 min at 4°C, and the supernatant was collected. The nuclear extracts were stored at -80°C until used. For western blot analysis, protein samples (50 µg per lane) were resolved using 4-12% SDS-PAGE (Life Technologies), and transferred to nitrocellulose membranes, and incubated overnight with anti-NRF2 (Cell Signaling Technology, Danvers, MA) and anti-OGG1 (Origene, Rockville, MD).
Lamin (Bethyl, Montgomery, TX, USA) was used as loading control. After incubation with an HRP-conjugated secondary IgG (Sigma), the blots were developed using the ECL detection system (Pierce Biotechnology, Rockford, IL, USA). Band intensities were visualized by ChemiDoc using the Quantity One software (BioRad, Hercules, CA).
Lamin (Bethyl, Montgomery, TX, USA) was used as loading control. After incubation with an HRP-conjugated secondary IgG (Sigma), the blots were developed using the ECL detection system (Pierce Biotechnology, Rockford, IL, USA). Band intensities were visualized by ChemiDoc using the Quantity One software (BioRad, Hercules, CA).
9
Western Blot Analysis of Protein Targets
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Western blot analysis of protein targets was performed as previously described (Tai et al. 2002 (link)(Tai et al. , 2007 (link)(Tai et al. , 2009)) (link). Briefly, 50 mg of total protein per sample was resolved on 12% SDSpolyacrylamide gels and subsequently transferred to PVDF membranes (BioRad) in transfer buffer (48 mM Tris, 39 mM glycine, 0.037% SDS, 20% methanol) at 100V for 1 h. Transfer and equal loading of protein were verified by Ponceau S stain (Sigma). Membranes were incubated for 1h at 48C in blocking solution containing 5% skim milk in Tris-buffered saline with Tween-20 (TBST; 10 mM Tris-HCl, 150mM NaCl, 0.05% Tween-20), then rinsed in TBST (3!10 min) before overnight incubation with primary antibodies specific for rat PNMT (Immunostar, Hudson, WI, USA; 1:3000 dilution), Egr-1 (C-19, Santa Cruz Biotechnologies; 1:1000), Sp1 (PEP-2, Santa Cruz; 1:2500), GR (M-20, Santa Cruz; 1:2000), AP-2 (Millipore, Billerica, MA, USA, 1:2000) in 5% skim milk-TBST. Membranes were then rinsed in TBST (3!10 min) and incubated with HRP-conjugated secondary IgG (Sigma; 1:5000) in 5% skim milk-TBST for 1 h, followed by a final series of rinses in TBST (3!10 min). Proteins were then detected by enhanced chemiluminescence (Haan & Behrmann 2007) (link) and subsequent exposure to film (Pierce, Rockford, IL, USA).
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