The EV pellet (20 μL) precipitated from pooled plasma (13 samples) from each group using ExoQuick was added to 20 μL of modified Laemmli 2× sample buffer and kept at 100°C for 7 minutes as described previously.35 (link) The samples were then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to Immobilon polyvinylidene fluoride membrane (Millipore Corporation, Bedford, MA). Before incubation with primary antibodies and secondary antibody, the membrane was blocked with nonfat skim milk solution (powdered milk dissolved at a concentration of 5% in Tris buffer saline with 0.1% Tween 20). Immunoblotting was performed overnight at 4°C using antibody diluted in nonfat skim milk solution. Anti–ICAM-1 (G-5, diluted at 1/200), anti–tumor susceptibility gene 101 (TSG-101) (4A10, diluted at 1/200), and anti–DAP-3 (10/DAP3, diluted at 1/200) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), Abcam Inc. (Toronto, ON, Canada), and BD Transduction Laboratories (Mississauga, ON, Canada), respectively. Membranes were then washed twice in Tris buffer saline with Tween 20, incubated for 30 minutes with horseradish peroxidase-conjugated secondary anti-mouse antibody (Jackson ImmunoResearch, Mississauga, ON, Canada) at a dilution of 1/10,000, and revealed using a Luminata Forte Western horseradish peroxidase substrate from Millipore Corporation.
Anti mouse horseradish peroxidase conjugated secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Anti-mouse horseradish peroxidase-conjugated secondary antibody is a laboratory reagent used for detection and visualization purposes. The product consists of a secondary antibody that specifically binds to mouse primary antibodies, conjugated with the enzyme horseradish peroxidase. This conjugate can be used in various immunoassay techniques to detect and amplify the signal from mouse-derived primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ez ecl, by Sartorius (2 mentions)
Ponceau s solution, by Merck Group (2 mentions)
Anti securin, by Abcam (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Luminata forte western horseradish peroxidase substrate, by Merck Group (1 mentions)
Immobilon polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Trpc1, by Santa Cruz Biotechnology (1 mentions)
Stim1, by Santa Cruz Biotechnology (1 mentions)
Orai2, by Santa Cruz Biotechnology (1 mentions)
Orai1, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Coomassie protein assay, by Thermo Fisher Scientific (1 mentions)
His tag monoclonal antibody, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 gel, by Thermo Fisher Scientific (1 mentions)
Curix 60, by AGFA HealthCare (1 mentions)
Chemiluminescence blotting substrate, by Roche (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
10 protocols using anti mouse horseradish peroxidase conjugated secondary antibody
1
EV Protein Expression Analysis by Western Blot
2
Western Blot Analysis of Lung Proteins
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Total proteins from frozen left lung samples were isolated using a RIPA lysis buffer
(Thermo Fisher) supplemented with a Protease inhibitor tablet cocktail according to
manufacturer's instructions (Pierce™, Thermo Fisher). Protein concentrations were
calculated using DC Protein Assay (Bio-Rad Hercules, CA, USA) following the manufacture
instructions. Isolated proteins were stored at −80°C until use. Ten micrograms of protein
were resolved in an SDS-PAGE (10–12% gradient gels) followed by transferring of proteins
onto nitrocellulose membranes. After appropriate blocking (5% nonfat dried milk) the blots
were probed with primary antibodies for TRPC1 (Santa Cruz, Dallas, TX, USA, sc-133076),
TRPC6 (Santa Cruz, sc-515837), STIM1 (Santa Cruz, sc-166840), ORAI2 (Santa Cruz,
sc-376757), and ORAI1 (Santa Cruz, sc-377281), diluted 1:1000 in 5% powdered nonfat dry
milk overnight at 4°C. After washing, membranes were incubated with horseradish
peroxidase-conjugated secondary anti-mouse antibody (Jackson ImmunoResearch, West Grove,
PA, USA) diluted 1:5000 in 5% powdered nonfat dry milk. Levels of proteins were normalized
to the β-actin (Sigma-Aldrich, AC-74) content of the same sample. The signals obtained
were scanned with densitometry through a detection device by chemiluminescence (Odyssey
Imaging System LI-COR Biosciences, Lincoln, NE, USA) quantified with the Image J software
(NIH, Bethesda, MD, USA).
(Thermo Fisher) supplemented with a Protease inhibitor tablet cocktail according to
manufacturer's instructions (Pierce™, Thermo Fisher). Protein concentrations were
calculated using DC Protein Assay (Bio-Rad Hercules, CA, USA) following the manufacture
instructions. Isolated proteins were stored at −80°C until use. Ten micrograms of protein
were resolved in an SDS-PAGE (10–12% gradient gels) followed by transferring of proteins
onto nitrocellulose membranes. After appropriate blocking (5% nonfat dried milk) the blots
were probed with primary antibodies for TRPC1 (Santa Cruz, Dallas, TX, USA, sc-133076),
TRPC6 (Santa Cruz, sc-515837), STIM1 (Santa Cruz, sc-166840), ORAI2 (Santa Cruz,
sc-376757), and ORAI1 (Santa Cruz, sc-377281), diluted 1:1000 in 5% powdered nonfat dry
milk overnight at 4°C. After washing, membranes were incubated with horseradish
peroxidase-conjugated secondary anti-mouse antibody (Jackson ImmunoResearch, West Grove,
PA, USA) diluted 1:5000 in 5% powdered nonfat dry milk. Levels of proteins were normalized
to the β-actin (Sigma-Aldrich, AC-74) content of the same sample. The signals obtained
were scanned with densitometry through a detection device by chemiluminescence (Odyssey
Imaging System LI-COR Biosciences, Lincoln, NE, USA) quantified with the Image J software
(NIH, Bethesda, MD, USA).
3
Western Blot Analysis of 6xHis-tagged Proteins
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24h after transfection, cells were collected using trypsin (Gibco) and resuspended in Laemmli 1X buffer. Proteins were denatured at 100°C for 5 min. Soluble proteins were retrieved after centrifugation at 15000 g at 4°C for 10 minutes and quantified using the Coomassie protein assay (Thermo Scientific). 25 µg of proteins were separated on a NuPAGE 4% -12% gel (Invitrogen, Thermo Fisher Scientific) and transferred to an Optitran BA-S83 nitrocellulose membrane (GE Healthcare Life Science). The membrane was then blocked in PBS with 5% non-fat milk for 30 min, before being incubated with the primary antibody (6x-His Tag Monoclonal Antibody, Invitrogen) overnight at 4°C. After washing the membrane five times for 5 min with PBS, it was incubated for 30 min in PBS with 5% non-fat milk and then for 1h at RT with horseradish peroxidase-conjugated secondary anti-mouse antibody (1:10000 dilution in PBS with 5% non-fat milk, Jackson Immunoresearch Laboratories), and washed again. Proteins were detected with the chemiluminescence detection reagent Perkin Western Lightning plus ECL (Perkin Elmer) and visualized using a radiology film processor (Curix 60, AGFA).
4
Protein Expression Analysis in HEK-293T Cells
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Total protein was extracted from transiently-expressing HEK-293T cells with cell lysis buffer (0.1 M Tris-HCl pH 7.8, 0.5% Triton X-100) containing cOmplete Mini EDTA-free protease inhibitor (Roche). Concentration of protein extracts was determined using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories) according to the manufacturers’ instructions. Protein samples (∼250 μg) were resolved by sodium dodecylsulphate-polyacrylamide gel electrophoresis (7.5% polyacrylamide) and transferred to a polyvinylidene fluoride membrane at 70 mA for 20 hours at 4°C. The membrane was blocked using 5% skim milk in TBST before addition of the primary antibodies; 34C (Sigma) for detection of RyR1 or anti-α-tubulin (Sigma) as a loading control, followed by horseradish peroxidase-conjugated anti-mouse secondary antibody (Jackson laboratories). Proteins were visualized using an X-ray developer after brief exposure with chemiluminescence blotting substrate (Roche) according to the manufacturer’s instructions. Successful expression of RyR1 protein was assessed by visual inspection of the film image, comparing the RyR1 band with the matching tubulin band.
5
Immunoblotting of Helicobacter pylori Neutrophil-Activating Protein
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Immunoblotting was performed essentially the same as previously described [23 (link)]. SDS-PAGE-separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% nonfat milk in Tris-buffered saline/Tween-20 (TBST) containing 50 mM Tris‐HCl, pH 7.4, 15 mM NaCl, and 0.1% Tween‐20 at room temperature for 1 h. The membrane was then probed with hybridoma culture supernatants containing mouse monoclonal antibody MAb 16F4 against HP-NAP [26 (link)] with a dilution factor of 1:200 in TBST containing 5% bovine serum albumin (BSA) at 4°C overnight. The membrane was washed three times with 5% nonfat milk/TBST, for 10 min each time, and then probed with horseradish peroxidase-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) at a dilution of 1:5000 in 5% nonfat milk/TBST at room temperature for 1 h. After the membrane was washed three times with TBST for 10 min each, the signal of HP-NAP on the membrane was visualized by an enhanced chemiluminescence (ECL) system (PerkinElmer, Waltham, MA, USA) and detected by LAS-3000 imaging system (Fujifilm, Tokyo, Japan).
6
Quantifying Nascent Protein Translation
7
Quantifying Capsule Shedding in Cryptococcus
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The relative amount of capsule shedding in the cell supernatant was assayed as previously described [21 (link),30 (link)]. Briefly, capsule induced cultures (incubated as described above) were incubated at 70°C for 15 minutes to denature enzymes, after which the cells were pelleted and the supernatant was sterile filtered. This conditioned medium was then run on a 0.6% agarose gel for 15 hours at 25 V, followed by transfer to a positively charged nylon membrane using Southern blotting methods. The membrane was air dried overnight, followed by blocking with 5% skim milk in Tris-Buffered Saline-Tween-20 (TBST). To detect capsule polysaccharide, blots were incubated with a mouse monoclonal anti-GXM antibody, MAb18B7 (1 μg/ml) [92 (link),93 (link)] for 1 hour, washed 3x with TBST, and incubated with an anti-mouse horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) for 1 hour. Blots were washed 3x with TBST and capsule polysaccharide was detected by enhanced chemiluminescence (ECL Prime Western blotting detection reagent; GE Healthcare).
8
Western Blot Analysis of Securin
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Protein samples were mixed with x4 Laemmli buffer, denatured (10 min, 96 °C), and resolved on freshly made 10% acrylamide gel using a Tris-glycine running buffer. Proteins were then electro-transferred onto a nitrocellulose membrane (Bio-Rad; #162-0115) using Trans-Blot Turbo transfer system (Bio-Rad). Ponceau S Solution (Sigma-Aldrich; #81462) was used to verify transfer quality. Membrane was washed (TBS), blocked (5% skimmed milk in TBST), and incubated (RT, 1 h) with antibody solution (2.5% BSA and 0.05% sodium azide in PBS) before blotted with anti-Securin (Abcam; #AB3305) primary antibody (RT, 2 h). Anti-mouse Horseradish peroxidase-conjugated secondary antibody was purchased from Jackson ImmunoResearch (#115-035-003). ECL Signal was detected using EZ-ECL (Biological Industries; #20-500-171).
9
Western Blot Analysis of G6PD and TLR4 in DRGs
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Expressions of G6PD and TLR4 in L4-L6 DRGs were determined by western blotting analysis, as previously described in detail.18 (link),23 (link),25 (link) The antibodies in the present study included mouse anti-TLR4 (1:200, Santa Cruz Biotechnology, Inc.), mouse anti-G6PD (1:200, Santa Cruz Biotechnology, Inc.), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000, Goodhere Biotechnology Co., Ltd.), anti-mouse horseradish peroxidase-conjugated secondary antibody (1:2000; Jackson ImmunoResearch Laboratories, Inc.), and anti-rabbit peroxidase-conjugated secondary antibody (1:2000; Jackson ImmunoResearch Laboratories, Inc.).
10
Western Blot Analysis of Securin
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Protein samples were mixed with x4 Laemmli buffer, denatured (10 min, 96°C), and resolved on freshly made 10% acrylamide gel using a Tris-glycine running buffer. Proteins were then electro-transferred onto a nitrocellulose membrane (Bio-Rad; #162-0115) using Trans-Blot Turbo transfer system (Bio-Rad). Ponceau S Solution (Sigma-Aldrich; #81462) was used to verify transfer quality. Membrane was washed (TBS), blocked (5% skimmed milk in TBST), and incubated (RT, 1 hr) with antibody solution (2.5% BSA and 0.05% sodium azide in PBS) before blotted with anti-Securin (Abcam; #AB3305) primary antibody (RT, 2 hrs). Anti-mouse Horseradish peroxidase-conjugated secondary antibody was purchased from Jackson ImmunoResearch (#115-035-003). ECL Signal was detected using EZ-ECL (Biological Industries; #20-500-171).
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