Purified C3 fragments (C3b, iC3b, C3dg; 0.25 μg in PBS each) were subjected to electrophoresis (4–15% SDS-PAGE) under reducing and denaturing conditions. Proteins were transferred onto a polyvinylidene difluoride membrane and blocked by incubation for 1 h in PBS containing 5% nonfat dry milk). The membrane was incubated for 1 h at RT with mouse anti-iC3b mAb (1:5000). Following washes, the membrane was incubated with anti-mouse HRP-conjugated secondary antibody (1:2000, #170-6516, BioRad). Bands were developed with HRP substrate (Immobilon, Millipore) and images were acquired using a ChemiDoc system with ImageLabTM software (v5.1, BioRad).
Anti mouse hrp conjugated secondary antibody
Manufactured by Bio-Rad
Sourced in United States
Anti-mouse HRP-conjugated secondary antibody is a laboratory reagent used to detect the presence of mouse primary antibodies in various immunoassay applications. It consists of a secondary antibody that is conjugated to the enzyme horseradish peroxidase (HRP), which can catalyze a colorimetric or chemiluminescent reaction for signal amplification and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated anti rabbit secondary antibody, by Bio-Rad (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Hrp conjugated anti rabbit secondary antibody, by Abcam (2 mentions)
Arhgef39, by Bio-Rad (2 mentions)
Image lab 5, by Bio-Rad (2 mentions)
β actin, by Bio-Rad (2 mentions)
Alx 804 639 l001, by Abcam (2 mentions)
Mabe1016, by Merck Group (2 mentions)
Immobilon, by Merck Group (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Image labtm software, by Bio-Rad (1 mentions)
Colloidal coomassie blue, by Bio-Rad (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Anti penta his antibody, by Qiagen (1 mentions)
Luminata forte, by Merck Group (1 mentions)
A1978, by Merck Group (1 mentions)
Sc 74443, by Santa Cruz Biotechnology (1 mentions)
Supersignal west femto substrate, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
14 protocols using anti mouse hrp conjugated secondary antibody
1
Western Blot Analysis of C3 Fragments
2
Quantifying TLR4 Protein Dynamics
3
SDS-PAGE and Western Blot Analysis
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SDS PAGE analyses were performed on 4–20% gels stained with colloidal Coomassie blue (BioRad). For WB, proteins separated on SDS PAGE were transferred during 1 h on a PVDF membrane (Immobilon P, Millipore), blocked during 1 h in PBS containing 0.1% Tween 20 (PBST), 2.5% BSA (PBST/BSA) and probed (2 h at room temperature or overnight at 4 °C) with an anti-penta-HIS antibody (Qiagen) at 1/3000 dilution in PBST/BSA. The blot was washed 6 times during 5 min with PBST, then incubated for 2 h at room temperature with an anti-mouse HRP conjugated secondary antibody (BioRad) at 1/3000 dilution in PBST/BSA. After 6 washes in PBST, the blot was revealed using the ECL system (Luminata® forte, Millipore).
4
Protein Extraction and Western Blot Analysis
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After washing cells twice with ice-cold PBS, cells were incubated on ice for 15 min in protein lysis buffer containing 20 mM HEPES, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 2 mM Na3VO4, 10 mM NaF, and protease inhibitor cocktail (P8340, Sigma-Aldrich). Cells were harvested using a cell scraper, and cell lysates were prepared by centrifugation at 13,000 rpm for 15 min at 4 °C. Supernatant was collected and protein concentrations were determined by Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Samples were resolved by SDS-PAGE and transferred to PVDF membrane (EMD Millipore). After blocking membranes in 5% skim milk containing TBS-T (w/v) for 1 h at room temperature, membranes were incubated in primary antibodies (PTN, 1:500 dilution in 2% BSA containing TBS-T, sc-74443, Santa Cruz and β-actin, 1:2000 dilution in 2% BSA containing TBS-T, A1978, Sigma-Aldrich) overnight at 4 °C. Membranes were washed three times for 10 min in TBS-T and incubated in anti-mouse HRP-conjugated secondary antibody (1:3000 dilution in TBS-T, Bio-Rad, Hercules, CA, USA) for 1 h at room temperature. Following washing, membranes were developed using SuperSignal West Femto Substrate (Thermo Fisher Scientific) and scanned using ImageQuant LAS 4000 (GE Healthcare, Little Chalfont, UK).
5
BDNF Signaling Pathway Analysis
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Membranes were incubated for 1 h with blocking solution (5% milk in TBST) and then probed overnight at 4°C with mouse anti-BDNF (1:2000; Icosagen; 327-100 clone 3C11), rabbit anti-p75 (1:700; Alomone Labs; ANT-007), rabbit anti-TrkB (1:700; Alomone Labs; ANT-019), and rabbit anti-proBDNF (1:250; Abcam; ab72440) in TBST. β-III Tubulin was used as a loading control (1:2500; R&D Systems). Binding of primary antibodies was visualized with anti-mouse HRP-conjugated secondary antibody (1:3000; BIO-RAD) or anti-rabbit HRP-conjugated secondary antibody (1:3000; BIO-RAD). Membranes were developed using the ECL Plus Western blotting substrate (Thermo Fisher Scientific) for chemifluorescence with the Storm® Molecular Imager. Densitometry was carried out using ImageJ software (Schneider et al., 2012 (link)). The signal of each protein is expressed after subtraction of background signal and related to tubulin signal.
6
Cytochrome c Quantification in Thyroid Tissues
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Frozen thyroids have been weighted, homogenized and sonicated in lysis buffer (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 10 mM EDTA, 10% glycerol (v/v), 1% Triton X-100 (v/v), and protease inhibitors). Twenty-five micrograms of protein extracts, previously quantified with the Bradford assay (Bio-Rad, Hercules, CA), have been resolved by 15% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad). After blocking with 3% BSA (w/v) dissolved in TBS buffer/ 0.5% Tween-20 (v/v), membranes have been probed overnight at with anti-cytochrome c (mouse mAb, sc-13560; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-actin (mouse mAb, sc-47778; Santa Cruz Biotechnology) primary antibodies. Membranes have been then incubated for 1 h at room temperature with anti-mouse HRP-conjugated secondary antibody (Bio-Rad). All the experiments have been repeated trice. Blots have been acquired and processed using the ChemiDoc Imaging system (Bio-Rad). Cytochrome c quantification has been performed using the Image Lab software (version 6.0.1, Bio-Rad Laboratories). Results are shown as mean ± standard deviation (SD); the statistical significance of differences has been tested using the Student’s t test (GraphPad Prism, version 6.01).
7
Purification and Lipid-Binding Analysis of Sbf PH Domain
8
Mutation Analysis using Western Blot
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Primers carrying the mutations were designed using NEBaseChanger software.
Primer sequences: I80P-F: 5'-TTTCCTTATCCCATTTCTGGTGCAG-3', I80P-R: non-fat dry milk in Tris-buffer saline with 0.1% Triton X-100 (TBST) for 2 hours at room temperature. Then, the membranes were incubated with the primary antibodies in blocking solution overnight at 4°C. The following primary antibodies were used for the Western blotting: mouse antibodies against Flag (Sigma-Aldrich, St. Louis, MO, USA)
and mouse antibody against β-actin (Proteintech Group, Chicago, IL, USA). The primary antibodies were detected with anti-mouse HRP-conjugated secondary antibodies (1:5000; Bio-Rad, Hercules, CA, USA), and the signal was developed using Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific). The relative intensity of the immunoreactive bands was quantified using the gel analysis tool provided in the ImageJ software. Normalization of the proteins of interest was performed relative to β-actin. Mean fluorescence intensity was calculated for each group.
Primer sequences: I80P-F: 5'-TTTCCTTATCCCATTTCTGGTGCAG-3', I80P-R: non-fat dry milk in Tris-buffer saline with 0.1% Triton X-100 (TBST) for 2 hours at room temperature. Then, the membranes were incubated with the primary antibodies in blocking solution overnight at 4°C. The following primary antibodies were used for the Western blotting: mouse antibodies against Flag (Sigma-Aldrich, St. Louis, MO, USA)
and mouse antibody against β-actin (Proteintech Group, Chicago, IL, USA). The primary antibodies were detected with anti-mouse HRP-conjugated secondary antibodies (1:5000; Bio-Rad, Hercules, CA, USA), and the signal was developed using Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific). The relative intensity of the immunoreactive bands was quantified using the gel analysis tool provided in the ImageJ software. Normalization of the proteins of interest was performed relative to β-actin. Mean fluorescence intensity was calculated for each group.
9
Western Blot Analysis of ARHGEF39 Expression
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3.0 × 105 HEK293 cells were seeded in each well of a six-well
plate (60–70% confluence) 24 h prior to transfection of the
microRNA (miR-215) expression vector (4 μg). Forty-eight hours
post-transfection, cells were lysed in lysis buffer (0.1m Tris,
150 mm NaCl, 10 mm EDTA, 0.2% Triton
X-100, 1% PMSF, protease inhibitor cocktail) at 4 °C for
10 min and centrifuged at 10 000 g for 30 min
at 4 °C, allowing cell debris to be pelleted and discarded. Western
blotting was performed as described previously.41 (link) Proteins were detected using primary antibodies for
60 min at room temperature or at 4 °C overnight. Secondary
antibodies were applied for 30–60 min at room temperature. Antibodies
were used as follows: ARHGEF39 (rabbit polyclonal, catalogue #131551,
manufacturer NovoPro Bioscience, Shanghai, China) at 1/2000 concentration,
B-actin (mouse monoclonal, catalogue #A5441, manufacturer Sigma-Aldrich) at
1/2000 concentration, secondary anti-mouse HRP-conjugated antibody (goat,
catalogue #1706516, Bio-Rad Laboratories, Hercules, CA, USA) at 1/5000
concentration and secondary anti-rabbit HRP-conjugated antibody (donkey
polyclonal, catalogue #16284, Abcam, Cambridge, UK) at 1/5000
concentration. Densitometry was performed using the Imagelab 5.2.1 (Bio-Rad)
program to calculate arbitrary units reflecting relative protein expression levels
for ARHGEF39 and β-actin.
plate (60–70% confluence) 24 h prior to transfection of the
microRNA (miR-215) expression vector (4 μg). Forty-eight hours
post-transfection, cells were lysed in lysis buffer (0.1
150 m
X-100, 1% PMSF, protease inhibitor cocktail) at 4 °C for
10 min and centrifuged at 10 000 g for 30 min
at 4 °C, allowing cell debris to be pelleted and discarded. Western
blotting was performed as described previously.41 (link) Proteins were detected using primary antibodies for
60 min at room temperature or at 4 °C overnight. Secondary
antibodies were applied for 30–60 min at room temperature. Antibodies
were used as follows: ARHGEF39 (rabbit polyclonal, catalogue #131551,
manufacturer NovoPro Bioscience, Shanghai, China) at 1/2000 concentration,
B-actin (mouse monoclonal, catalogue #A5441, manufacturer Sigma-Aldrich) at
1/2000 concentration, secondary anti-mouse HRP-conjugated antibody (goat,
catalogue #1706516, Bio-Rad Laboratories, Hercules, CA, USA) at 1/5000
concentration and secondary anti-rabbit HRP-conjugated antibody (donkey
polyclonal, catalogue #16284, Abcam, Cambridge, UK) at 1/5000
concentration. Densitometry was performed using the Imagelab 5.2.1 (Bio-Rad)
program to calculate arbitrary units reflecting relative protein expression levels
for ARHGEF39 and β-actin.
10
MiRNA-215 Overexpression in HEK293 Cells
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