Proteins (20 μg) were separated on SDS-PAGE gels. Then, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). The PVDF membranes were then blocked with 5% BSA diluted in TBS for 1 h at room temperature. Primary antibodies against CXCL10 (R&D), RANKL (Abcam), Cadherin-11 (Invitrogen), mTOR (Abcam), phosphorylated mTOR (Abcam), CREB (Abcam), phosphorylated CREB (Abcam), AKT (Abcam), phosphorylated AKT (Abcam), PI3K (Abcam), phosphorylated PI3K (Abcam) and GAPDH (Abcam) were then added according to the manufacturers’ protocols. The samples were agitated at 4 °C overnight. HRP-conjugated rabbit anti-mouse IgG (Abcam), mouse anti-rabbit IgG (Abcam) and goat anti-mouse IgG (Abcam) secondary antibodies were added and incubated at room temperature for 2 h. Densitometric analysis was performed using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories).
Hrp conjugated rabbit anti mouse igg
Manufactured by Abcam
Sourced in United Kingdom
HRP-conjugated rabbit anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) antibodies. The antibody is conjugated to horseradish peroxidase (HRP), an enzyme that can be used for detection in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Triton x 100, by Merck Group (2 mentions)
Hrp western blot analysis kit, by EASYBIO (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Hrp conjugated goat anti rabbit igg, by Abcam (2 mentions)
Cadherin 11, by Thermo Fisher Scientific (1 mentions)
Phosphorylated creb, by Abcam (1 mentions)
Phosphorylated akt, by Abcam (1 mentions)
Phosphorylated pi3k, by Abcam (1 mentions)
Rabbit anti mouse igg, by Abcam (1 mentions)
Cxcl10, by R&D Systems (1 mentions)
Gapdh, by Abcam (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Goat anti mouse igg, by Abcam (1 mentions)
Rankl, by Abcam (1 mentions)
Enhanced chemiluminescence method, by Beyotime (1 mentions)
Ab106066, by Abcam (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ab8245, by Abcam (1 mentions)
18 protocols using hrp conjugated rabbit anti mouse igg
1
Western Blot Analysis of Signaling Proteins
2
Quantitative Protein Analysis by Western Blot
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Total protein was extracted from tissues and cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration was determined by the BCA assay kit (Beyotime, Haimen, Jiangsu, P.R. China). The same amount of protein (50 μg) was separated using 10% SDS-PAGE gel, and then the protein was transfected to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with fat-free milk and incubated with antibody against GATA6 (1:1,000; ab106066; Abcam, Cambridge, MA, USA) and GAPDH (1:1,000; ab8245; Abcam) at 4°C overnight. Finally, the membrane was incubated with HRP-conjugated rabbit anti-mouse IgG (1:5,000; ab6728; Abcam). The protein was detected with the enhanced chemiluminescence method (Beyotime). Protein signals were quantified using Quantity One Software (Bio-Rad, Hercules, CA, USA).
3
Deglycosylation of Erythropoietin
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Denaturation of the protein by preheating the sample in denaturing buffer at 100°C for 10 min was carried out. To remove N-linked carbohydrates, each 20 μl of the reaction mixture was prepared with 20 ng of hEPO, 500 U of PNGase F, 2 μl of 10X GlycoBuffer 2, and 10% NP 40. To remove the O-linked carbohydrates, the samples were treated with 40,000 U of O-glycosidase and 50 U of neuraminidase. To take out both N-linked and O-linked carbohydrates, the same amount of hEPO sample was treated with 500 U of PNGase F, 40,000 U of O-glycosidase and 50 U of neuraminidase. All materials including enzymes and buffers were purchased from New England Biolabs (Ipswich, MA, USA). After incubation at 37°C for 4 hours, the samples underwent electrophoresis were transferred to a nitrocellulose membrane. After 1 h of blocking in TBS with 0.03% Tween-20 (MTBST) and 5% skim milk, the membrane was incubated with a mouse monoclonal anti-hEPO (R&D Systems, Minneapolis, USA) in MTBST (1/5,000 dilution) for 16 h. Following washing with TBST alone three times, the membrane was incubated with HRP-conjugated rabbit anti-mouse IgG (Abcam, Cambridge, UK) in MTBST (1/15,000 dilution) for 1 h. After washing three times with TBST, the detection of chemiluminescence was done by adding West Dura Extended Duration substrate (Thermo Fisher Scientific, MA, USA).
4
Western Blot Analysis of Subchondral Bone Proteins
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At sacrifice, samples of the right tibial subchondral bone were collected for western blot analysis. For coronin 1A (CORO1A), S100 calcium-binding protein A8 (S100A8) and TCIRG1 protein expression studies, 100 µg total protein from tibial subchondral bone was resolved using a 4-12% SDS-PAGE gel. After transfer to PVDF membranes, the membranes were blocked for 1 h at room temperature in 1X PBS containing 0.15% Tween-20 (1X PBST) and 5% non-fat dry milk, and subsequently immunoblotted with the indicated primary antibodies in 1X PBST with 5% non-fat dry milk at 4˚C overnight. The primary antibodies included anti-CORO1A (Aviva Systems Biology, Corp; cat. no. ARP38899_T100; 1:400), anti-S100A8 (Abcam; cat. no. ab92331; 1:1,000) and anti-TCIRG1 (Santa Cruz Biotechnology, Inc.; cat. no. sc-162300; 1:500). Signals were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) and horseradish peroxidase HRP-conjugated goat anti-rabbit IgG (Abcam; cat. no. ab97051; 1:5,000), HRP-conjugated rabbit anti-mouse IgG (Abcam; cat. no. ab6728; 1:5,000) and HRP-conjugated rabbit anti-goat IgG (Abcam; cat. no. ab6741; 1:5,000) secondary antibodies for 2 h at room temperature. β-actin (Abcam; cat. no. ab6276; 1:1,000) was used as the internal control.
5
ELISA for Virus Antigen Detection
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Viruses (H5, H1N1, H7N7) and chicken uninfected eggs fluid were diluted in coating buffer (bicarbonate/carbonate 100 mM, pH 9.6) to obtain the titer at 1000 HAU/mL. Then, 100 μL of each virus was coated on a 96-well microtiter plate (Greiner GmbH, Pleidelsheim, Germany) and incubated at 4 °C, overnight. After incubation, 200 μL phosphate buffered saline (PBS) plus 0.1% Tween 20 (PBS-T, pH 7.4) was used for washing. Following 5% non-fat dry milk blocking at 37 °C for 2 h, 100 μL candidate antibody (20 µg/mL) or cell supernatant was added to each well. After incubation for 1 h at 37 °C, the 96-well plate was washed with PBS-T before adding 100 μL horse-radish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (Catalog Number: ab97046, Abcam, Cambridge, UK; 0.02 μg/well) to detect antigens. After washing the plate five times with PBS-T (pH 7.4) to remove nonspecific binding, 100 μL 3,3′,5,5′-tetra methyl benzamine substrate solution (Invitrogen, Carlsbad, CA, USA) was added for color development. The sample was covered with foil for 10 min and the reaction was stopped by adding 100 μL 0.18 M sulfuric acid to each well. Optical density (OD) was determined on a microplate reader at 450 nm (SpectraMax® M Series Multi-Mode Microplate, San Jose, CA 95134, USA).
6
Measurement of Anti-Influenza B Antibodies
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Sera were collected at indicated times to analyze Ab response. ELISA was used to determine the serum Ab titers against B-NP Ag. The B/Yamagata/16/88 was split with 0.5% Triton X-100 (Sigma, St. Louis, MO, USA), and 96F MaxiSorp Nunc-Immuno plates were coated with the split virus (3,600 pfu/well). The serum samples were diluted in 1% non-fat milk PBS and 0.05% Tween 20 and incubated for 2 h at room temperature (RT). After washing, we added HRP-conjugated rabbit anti-mouse IgG (Abcam) or HRP-conjugated goat-anti mouse IgA (Zymed Laboratories Inc., San Francisco, CA, USA) as a secondary Ab and incubated for 1 h at RT. The samples were then incubated with a 3,3′,5,5′-tetramethylbenzidine substrate and solution. The sample reaction was stopped with 1M phosphoric acid and detected at 450 nm by Thermo Multiskan EX (Vantaa, Finland).
7
Western Blot Analysis of Viral Proteins
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Equal gc numbers of purified virus stocks were denatured by boiling in Laemmli sample buffer with 2-mercaptoethanol (Bio-Rad, Hercules, CA, USA) and electrophoresed on precast 4%–15% SDS-PAGE gels (Bio-Rad). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and reacted with anti-gD (DL6, Santa Cruz Biotechnology, Dallas, TX, USA) or anti-VP16 (Santa Cruz Biotechnology) antibody prior to incubation with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse immunoglobulin G (IgG; Abcam, Cambridge, UK). Membranes were developed with ECL (enhanced chemiluminescence) Plus (Thermo Fisher Scientific). Signal intensities were measured with ImageJ software.37 (link)
For screening of the B78-vIII clones, 107 cells of each clone were collected and lysed in 1 mL of 1× radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific). Protein denaturation, electrophoresis, and transfer to PVDF membranes were as above. Membranes were sequentially reacted with anti-EGFR monoclonal antibody H11 (Thermo Fisher Scientific) and HRP-conjugated rabbit anti-mouse IgG (Abcam, Cambridge, UK) and developed with ECL Plus.
For screening of the B78-vIII clones, 107 cells of each clone were collected and lysed in 1 mL of 1× radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific). Protein denaturation, electrophoresis, and transfer to PVDF membranes were as above. Membranes were sequentially reacted with anti-EGFR monoclonal antibody H11 (Thermo Fisher Scientific) and HRP-conjugated rabbit anti-mouse IgG (Abcam, Cambridge, UK) and developed with ECL Plus.
8
Recombinant Human TNF-α Production
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Restriction enzymes, T4 DNA ligase, and Phusion High-Fidelity DNA Polymerase were purchased from New England Biolabs (Ipswich, MA). Oligonucleotides were synthesized by Beijing AuGCT Biological Technology Co., Ltd (Beijing, China). The vector pET-22b and E. coli strain BL21 (DE3) were purchased from Novagen (San Diego, CA). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was from Sigma-Aldrich (St. Louis, MO). Sephacryl S-300 was from GE Healthcare Life Sciences (Uppsala, Sweden). Mouse anti-human TNF-α monoclonal antibody and HRP-conjugated rabbit anti-mouse IgG were from Abcam (UK, Cambridge). Standard recombinant hTNF-α was purchased from National Institutes for Food and Drug Control (Beijing, China). The fermentor was from B. Braun (Germany). All other reagents were of analytical grade.
9
Verification of Vaccine Antigens by Western Blot
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The UreB-HspA expressed by the vaccine strain SH02 and the rUreB-HspA expressed and purified from E. coli were verified by Western Blot analysis. The vector strain without antigen expression, SH02, purified rUreB, rHspA, and rUreB-HspA were denatured and separated by 15% SDS-PAGE, transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Mannheim, Germany), and then blocked with PBST containing 5% skimmed milk at 37°C for one hour to prevent non-specific protein binding. The membranes were incubated with appropriate dilution of UreB monoclonal antibodies (21 (link)) or immunized mice sera at 37°C for one hour, washed with PBST three times, and then incubated with HRP-conjugated rabbit anti-mouse IgG (1:5000 dilution, Abcam, Cambridge, UK) at 37°C for one hour. The HRP Western Blot Analysis Kit (Easybio, Beijing, China) was used to detect the binding reaction.
10
Expression and Validation of B/Yamagata NP Protein
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NP gene cDNA from the B/Yamagata/16/88 virus (15 (link)) was generated by PCR using a forward primer that included a NheI restriction enzyme site and Kozak sequence to enhance translation (5′-CGGCTAGCGCCACCATGTCCAACATGGATATT-3′) and a reverse primer (5′-GCTCGAGTCATTAATAGTCGAGGTCATC-3′) that included 2 stop codons and an XhoI restriction enzyme site. We cloned DNA from PCR reactions into the pGX27 vector (16 (link)) through NheI/XhoI double digestion, resulting in pGX27/B-NP. To validate immunogen expression, HEK293 cells were transfected with 8 µg of pGX27/B-NP or pGX27 with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), used according to the manufacturer's directions. As described previously, rAd/B-NP was a positive control (15 (link)). The transfected cells were harvested after 48 h, and the resulting cell lysate and supernatant were subjected to SDS-PAGE and Western blotting using a mouse polyclonal B/Yamagata-specific antiserum and HRP—conjugated rabbit anti-mouse IgG (Abcam, Cambridge, UK) as a secondary Ab.
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