The final amounts of unmodified and citrullinated rhPAD4 proteins were adjusted to 200 ng/well. Unmodified and citrullinated rhPAD4 were resolved by 10% polyacrylamide gel electrophoresis, then blotted onto nitrocellulose membranes. Non-specific protein binding was blocked for 1 h at approximately 25 °C using 5% non-fat dried milk in Tris-buffered saline containing 0.1% Tween-20. Unmodified rhPAD4 was detected using a primary mouse anti-PAD4 monoclonal antibody (Merck KGaA, Darmstadt, Germany) diluted 1:2000, and secondary horseradish peroxidase (HRP)-linked anti-mouse IgG (Cell Signaling Technology, Danvers, MA, USA), diluted 1:1000. Citrullinated rhPAD4 on blots was chemically modified overnight at 37 °C to enable the detection of citrulline residues using human anti-modified citrulline antibody (Merck) as described by the manufacturer22 (link), and then probed with 1:1000-diluted human anti-modified citrulline antibody for 1 h at approximately 25 °C. Bound antibodies were detected using 1:10,000-diluted HRP-conjugated goat anti-human IgG (Merck), then chemiluminescence was measured.
Horseradish peroxidase hrp linked anti mouse igg
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase (HRP)-linked anti-mouse IgG is a secondary antibody reagent used in various immunoassay techniques. It consists of an HRP enzyme conjugated to an antibody that specifically binds to mouse immunoglobulin G (IgG). This reagent is designed to detect and signal the presence of mouse IgG in samples.
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Lab products found in correlation
High binding 96 well elisa plates, by Greiner (2 mentions)
Anti rabbit igg, by Cell Signaling Technology (2 mentions)
Hrp conjugated goat anti human igg, by Merck Group (1 mentions)
Elisa, by Agilent Technologies (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Complete ultra, by Roche (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Hrp linked anti rabbit igg, by Cell Signaling Technology (1 mentions)
Ecl select reagent, by GE Healthcare (1 mentions)
Bradford dye binding method, by Bio-Rad (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Phosstop, by Roche (1 mentions)
Phosphatase inhibitor, by Abcam (1 mentions)
Femtolucent plus hrp, by G Biosciences (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti p65, by Santa Cruz Biotechnology (1 mentions)
Anti p53, by Santa Cruz Biotechnology (1 mentions)
7 protocols using horseradish peroxidase hrp linked anti mouse igg
1
Western Blot Detection of Citrullinated rhPAD4
2
Quantification of Antigen-Specific IgG by ELISA
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To measure antigen-specific IgG, ELISAs were performed, as described previously (29 (link)). High-binding 96-well ELISA plates (Greiner Bio-one, Kremsmünster, Austria) were coated overnight at 4°C with 1 μg/ml recombinant protein (produced as described elsewhere (12 (link), 18 (link), 19 (link), 21 (link))), washed with PBS–Tween (phosphate-buffered saline–0.05% Tween) and incubated with blocking buffer (1% BSA in PBS, pH 6.5) for 2 h at room temperature. Mouse sera (collected at day 42 before tick challenge) were diluted in blocking buffer, added to the wells and incubated for 1 h at room temperature. Plates were washed and incubated for 1 h with horseradish peroxidase (HRP)-linked anti-mouse IgG (Cell Signaling, Beverly, MA, USA) diluted 1:1,000 in blocking buffer. The plates were washed again and developed using TMB substrate [50 µl TMB chromogene in 5 ml TMB substrate buffer (8,2 gr NaAc and 21 gr citric acid monohydrate dissolved in 1 liter H2O + 10 µl 3% H2O2, pH 5)] and optical density was measured in a Biotek (Winooski, VT, USA) ELISA plate reader at 450–655 nm.
3
Phospho-ERK Levels in ISOS-1 Cells
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Phosphorylated extracellular signal-regulated kinase (phospho-ERK) levels in ISOS-1 cells incubated with 100 ng/ml wild-type FGF1 or 100 ng/ml FGF1-PIGN alone or with 1 μM PD0325901 for 1, 12 and 24 h were examined by western blot analysis. Cells were washed with phosphate-buffered saline (PBS) then lysed with lysis buffer (50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, phosphatase inhibitor [PhosSTOP] [Roche Diagnostics] and protease inhibitors [cOmplete ULTRA] [Roche Diagnostics]). The lysates were collected using a cell scraper and protein concentration evaluated using the Bradford dye-binding method (Bio-Rad Protein Assay) (Bio-Rad Laboratories, Hercules, CA). Ten micrograms of protein lysate from each sample were separated on a Mini-PROTEAN TGX gel (Bio-Rad Laboratories) and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA). After blocking with bovine serum albumin (BSA) (Thermo Fisher Scientific), membranes were incubated with primary antibodies against ERK (Cell Signaling Technology, Danvers, MA), phospho-ERK (Cell Signaling Technology) and β-actin (Cell Signaling Technology) at 4°C overnight. Then, membranes were incubated with HRP-linked anti-rabbit IgG (Cell Signaling Technology) or horseradish peroxidase (HRP)-linked anti-mouse IgG (Cell Signaling Technology), washed and developed with ECL Select reagents (GE Healthcare).
4
Western Blot Analysis of WJ-MSCs
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Following different experimental treatments, WJ-MSCs were lysed in RIPA buffer containing protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor (Abcam, Cambridge, UK). Protein concentration was quantified using the Bradford assay. Western blotting was performed as per standard protocols. The primary and secondary antibodies used were as follows: anti-VTN, anti-p53, anti- p65, anti-GAPDH (all from Santa Cruz Biotechnology, Inc. Dallas, TX, USA), and horseradish peroxidase (HRP)-linked anti-mouse IgG (Cell Signaling Technology, Inc., Danvers, MA, USA). Immunoreactivity was detected using chemiluminescent femtoLucent PLUS-HRP (G-Biosciences, Maryland Heights, MO, USA). Chemiluminescent blots were imaged using G:Box (Syngene, Frederick, MD, USA).
5
IgG Quantification Against Borrelia BB0405
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To measure IgG directed against BB0405, ELISAs were performed according to previous described protocol10 (link). High-binding 96-well ELISA plates (Greiner Bio-one, Kremsmünster, Austria) were coated overnight at 4 °C with 1 μg/ml recBB0405, washed with PBS–Tween (phosphate-buffered saline–0.05% Tween) and incubated with blocking buffer (1% BSA in PBS) for 2 h at room temperature. Mouse sera (collected at day 42 before tick challenge) were diluted in blocking buffer, added to the wells and incubated for 1 h at room temperature. Plates were washed and incubated for 1 h with horseradish peroxidase (HRP)-linked anti-mouse IgG (Cell Signaling, Beverly, MA, USA) diluted 1:1000 in blocking buffer. The plates were washed again and developed using TMB substrate (50 μl TMB chromogene in 5 ml TMB substrate buffer (8.2 g NaAc and 21 gr citric acid monohydrate dissolved in 1 L H2O + 10 μl 3% H2O2) and optical density was measured in a Biotek (Winooski, VT, USA) ELISA plate reader at 450–655 nm.
6
Antibody Characterization for TGF-β Pathway
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Anti-fibronectin rabbit polyclonal antibody (ab2413) and anti-CD31 rabbit recombinant multiclonal antibody (ab281583) were purchased from Abcam (Cambridge, MA); SMAD2 (L16D3) mouse monoclonal antibody (# 3103), phospho-SMAD2 (138D4) rabbit monoclonal antibody (# 3108), SMAD3 (C67H9) rabbit monoclonal antibody (# 9523), phospho-SMAD3 (C25A9) rabbit monoclonal antibody (# 9520), SMAD4 (D3M6U) rabbit monoclonal antibody (# 38454), horseradish peroxidase (HRP)-linked anti-mouse IgG (# 7076) and anti-rabbit IgG (# 7074) were purchased from Cell Signaling (Beverly, MA); mouse monoclonal antibody HLA-G was purchased from EXBIO (#11-499-C100) ; cytokeratin 7-specific rabbit polyclonal antibody (# 17513-1-AP) was purchased from Proteintech (Wuhan, China); mouse monoclonal anti-α-Tubulin antibody (sc-23948) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); mouse monoclonal anti-cytokeratin 8 antibody (GB12233-100) was obtained from Servicebio (Wuhan, China); anti-Mouse Secondary Antibody, Alexa Fluor 488 (#A11001), anti-Rabbit Secondary Antibody, Alexa Fluor 594 (# A11012) and ACVR1B (ALK4) TaqMan primers (# 433182) were obtained from Thermo Fisher (Waltham, MA); recombinant human/mouse/rat activin A protein was provided by R&D (Minneapolis, MN); SB431542 (TGF-beta type I receptor inhibitor) (# S4317) was purchased from Sigma Aldrich (St. Louis, MO).
7
Comprehensive Western Blot Analysis
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For Western blot, anti-DGKA (ab197249) was purchased from Abcam. Anti-GAPDH (#5174), anti-b-Actin (#4970), anti-pAKT-Ser473 (#4070), anti-pAKT-Thr308 (#13038), anti-pERK1/2-Thr202/Tyr204 (#4370), anti-AKT (#4691), anti-ERK1/2 (#4695), anti-CDK4 (#12790), anti-CDK6 (#13331), anti-Cyclin D1 (#2978), anti-Cyclin D2 (#3741), horseradish peroxidase (HRP)-linked antimouse IgG (#7076), and anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology.
Cells were lysed in RIPA lysis buffer containing 1 mmol/L phenylmethylsulfonyl fluoride (Beyotime). Lysates were clarified by centrifugation at 10,000 Â g for 10 minutes at 4 C. Protein concentration was measured using the BCA Assay (Thermo Scientific) and diluted in sample loading buffer (Life Technologies). Protein (15 mg) was loaded per well and separated by electrophoresis (Bio-Rad) in a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Protein was electrophoretically transferred to a PVDF membrane (Amersham Pharmacia Biotech), incubated with a blocking solution of 5% BSA at room temperature for 1 hour, and incubated with primary antibodies prepared in 2% BSA at 4 C overnight. On day 2, the PVDF membrane was washed in 0.1% TBST buffer and incubated with HRP-linked secondary antibodies. The protein bands were detected by enhanced chemiluminescence.
Cells were lysed in RIPA lysis buffer containing 1 mmol/L phenylmethylsulfonyl fluoride (Beyotime). Lysates were clarified by centrifugation at 10,000 Â g for 10 minutes at 4 C. Protein concentration was measured using the BCA Assay (Thermo Scientific) and diluted in sample loading buffer (Life Technologies). Protein (15 mg) was loaded per well and separated by electrophoresis (Bio-Rad) in a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Protein was electrophoretically transferred to a PVDF membrane (Amersham Pharmacia Biotech), incubated with a blocking solution of 5% BSA at room temperature for 1 hour, and incubated with primary antibodies prepared in 2% BSA at 4 C overnight. On day 2, the PVDF membrane was washed in 0.1% TBST buffer and incubated with HRP-linked secondary antibodies. The protein bands were detected by enhanced chemiluminescence.
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