Brain tissue was homogenized in 5% glucose in distilled water in grinding tubes (Bio-Rad) and adjusted to 10% (w/v) by using a TeSeETM Precess 48TM homogenizer (Bio-Rad) following the manufacturer’s instructions. To determine the presence of PrPres in transgenic mouse brains, 100 µL of 10% brain homogenate were analyzed by Western blotting as previously described [7 (link)] with some modifications. Briefly, digestion was done with 40 µg/mL of proteinase K in buffer 5% sarkosyl, 5% Triton X100, 1 M Urea and 16 mM Tris–HCl (pH 9.6) at 60 °C for 15 min. Samples were electrophoresed in 12% Criterion XT Bis–Tris Gel (BioRad). For immunoblotting, membranes were incubated with 12B2 PrP monoclonal antibody (epitope 93WGQGG97 of the sheep PrP sequence) at a final concentration of 1 μg/mL. Immunocomplexes were detected with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Pharmacia Biotech) after incubating the membranes for 1 h, and blots were developed with chemiluminescent substrate ECL Select (GE Healthcare Amersham Biosciences). Images were captured using ChemiDoc XRS + System and then processed using Image Lab 5.2.1 Software.
Horseradish peroxidase conjugated anti mouse igg
Manufactured by Cytiva
Sourced in Sweden, Japan, United Kingdom
Horseradish peroxidase-conjugated anti-mouse IgG is a secondary antibody used in various immunoassays and immunodetection techniques. It binds to mouse immunoglobulin G (IgG) and is conjugated with the enzyme horseradish peroxidase, which can be used to generate a colorimetric or chemiluminescent signal for detection purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ecl select, by Cytiva (6 mentions)
Tesee precess 48tm homogenizer, by Bio-Rad (4 mentions)
Grinding tubes, by Bio-Rad (4 mentions)
Image lab 5, by Bio-Rad (3 mentions)
Tesee, by Bio-Rad (2 mentions)
Chemidoc wrs system, by Bio-Rad (2 mentions)
Complete inhibitor cocktail, by Roche (2 mentions)
Criterion xt bis tris gel, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Enhanced chemiluminescence detection kit, by Cytiva (1 mentions)
Ecl western blotting detection system, by Cytiva (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Image lab software v5, by Bio-Rad (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
16 protocols using horseradish peroxidase conjugated anti mouse igg
1
Western Blot Analysis of PrP^res in Transgenic Mouse Brains
2
Detecting PrP^res in Transgenic Mouse Brains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brain tissue was homogenized in 5% glucose in distilled water in grinding tubes (Bio-Rad) and adjusted to 10% (w/v) by using a TeSeE™ Precess 48TM homogenizer (Bio-Rad) following the manufacturer’s instructions. To determine the presence of PrPres in transgenic mouse brains, 100 μL of 10% brain homogenate were analyzed by WB as previously described14 (link). For immunoblotting, membranes were incubated with Sha31 PrP monoclonal antibody (epitope 148-YEDRYYRE-155 of the goat PrP sequence)26 (link) at a final concentration of 1 μg/mL. Immunocomplexes were detected with horseradish peroxidase- conjugated anti-mouse IgG (Amersham Pharmacia Biotech) and blots were developed with chemiluminescent substrate ECL Select (GE Healthcare Amersham Biosciences). Images were captured using ChemiDoc XRS + System and then processed using Image Lab 5.2.1 Software.
3
Quantifying Phosphorylated PKB in Human Islet Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
About 100 human islets were lysed in 30 μL lysis buffer containing 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulphate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mmol/L PMSF and 10 μg/ml aprotinin for 25 min on ice and vortexed every 5 min. Samples were centrifuged (15,000 g, 10 min, 4°C) and the supernatant fractions were frozen at − 70°C until analyzed. Aliquots of protein (15μg) from islet lysates were electrophoresed on polyacrylamide gel, then incubated for 1 h at room temperature with rabbit anti-phospho-PKB (1:750; Cell Signaling) which detects Ser473 phosphorylated PKB (or its equivalent sites on PKBβ-Ser474 and PKBγ-Ser472) or rabbit anti-total PKB (1:750; Cell Signaling) which detects all three forms of PKB. Membranes were then washed and incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:5000; Amersham, Baie D’Urfe, QC, CA) for 1 h. Immunodetection was performed using an enhanced chemiluminescence detection kit (Amersham). Protein bands on the films were analyzed by densitometry using Image Lab software (Bio-Rad, Mississauga, ON, CA).
4
Hippocampus and Cortex PrPC Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Homogenized hippocampi and prefrontal cortices (ca.30 μg of total proteins) were subjected to sodium-dodecyl sulphate 10% or 15% SDS-PAGE. Two different western blots were probed with anti-PrP SAF32 (Spi-Bio, Bertin Pharma, France) or anti-PrP SAF61 (Spi-Bio, Bertin Pharma, France) which recognized PrPC. Bound antibodies were visualized with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Biosciences, Uppsala, Sweden) and immunoreactivity assessed by chemiluminescence reaction using the ECL Western blotting detection system (Amersham, UK). PrPC bands were subjected to densitometric scanning analysis, performed by Mac OS X (Apple Computer International), using NIH Image 1.62 software. ImageJ densitometry software (Version 1.6, National Institutes of Health, Bethesda, MD) was used for band quantitative densitometric analysis. Selected bands were quantified on the basis of their relative intensities, which are reported as arbitrary densitometric units. The software allows the measurement of density profiles, peak heights as well as peak intensity (average OD of the band, INT) or volume (average OD of the band times its area, INT*mm2) of the band.
5
Western Blot Analysis of Phosphorylated ERK
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Extracts of HeLa cells were prepared by adding 200 μl of SDS-PAGE sample buffer (50 mM Tris HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1 M dithiothreitol, 1% bromophenol blue) to cells in each well of a 6-well plate, scraping off the cells, and sonicating the suspension. A 10 μl aliquot of extract was loaded onto each well of an SDS polyacrylamide gel, which was electrophoresed and transferred to a PVDF filter (Amersham, Buckinghamshire, UK), and each filter was incubated with mouse monoclonal antibody against phosphorylated extracellular signal-regulated kinase, ERK (2 μg/ml, Sigma) or rabbit polyclonal anti-ERK antibody (0.6 μg/ml, Sigma). Horseradish peroxidase-conjugated anti-mouse IgG (Amersham) and ECL Plus (Amersham) were used to detect all bound primary antibodies. Chemiluminescence was captured as digital images, using a CCD camera, AE-6972FC (Atto, Tokyo, Japan), and total intensities of the two bands corresponding to ERK1 and ERK2, were quantified using Photoshop (Adobe systems, San Jose, CA).
6
Western Blot for Total PrP and PrPres
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For total PrP detection, 15 µl of the different samples were analyzed by WB. Samples were prepared as previously detailed52 (link). Serial dilutions were done by diluting the original sample into loading buffer just before electrophoresis. For immunoblotting, membranes were incubated with Sha31 mAb19 (link) that recognizes 144-WEDRYYRE-151 epitope of the mouse-PrP sequence. To determine the presence of PrPres, 20 μl of the different samples were analyzed by WB as previously described53 (link). For immunoblotting, membranes were incubated with Sha31 mAb. Immunocomplexes were detected with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Pharmacia Biotech) after incubating the membranes for 1 hour. Immunoreactivity was visualized by chemiluminescence with ECL Select (GE Healthcare Amersham Biosciences). When necessary, WB images were quantified using ImageLab software v5.2.1(BioRad).
7
Cadherin Expression and Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMP7 was from R&D systems (Minneapolis, MI). Antibodies against phosphorylated p38 (P‐p38) and phosphorylated ERK (P‐ERK) were from Cell Signaling Technology (Beverly, MA). Anti‐p38 antibody (C‐20) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti‐ERK antibody (Erk1/2‐CT, rabbit polyclonal IgG) was from Upstate Biotechnology (Lake Placid, NY). Mouse and rabbit anti‐cadherin‐11 antibodies were from Zymed Laboratories (South San Francisco, CA). Anti‐uvomorulin/E‐cadherin antibody was from Sigma (Saint Louis, MO), and anti‐K‐cadherin antibody (cadherin 6, sc‐31024, sc‐59974) was from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase‐conjugated anti‐mouse IgG and anti‐rabbit IgG were from Amersham (Buckinghamshire, UK). A p38 inhibitor SB203580 and a MEK inhibitor PD98059 were from Carbiochem‐Novabiochem (La Jolla, CA). DMEM, FBS, penicillin, streptomycin, Hanks' balanced salt solution, and trypsin‐EDTA were from Gibco Laboratories (Grand Island, NY).
8
Prion Protein Immunodetection Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse anti-PrP SAF32 monoclonal antibody (Spi-Bio, Bertin Pharma, France) and mouse anti-PrP SAF61 monoclonal antibody (Spi-Bio, Bertin Pharma, France) which recognized prion protein at different epitopes (SAF32 a.a. 79–92; SAF 61 a.a.144–160), goat anti-PrP C-20 polyclonal antibody or mouse anti-Thy-1 monoclonal antibody (Abcam, Cambridge, UK) were employed. Bound antibodies were visualized with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Biosciences, Uppsala, Sweden) or anti-goat IgG (Sigma-Aldrich, Milan, Italy)
9
Western Blot Analysis of TXNIP and TRX
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from untreated normal skin, normal skin treated with 25 mM D-allose for 2 weeks, untreated tumor tissue, and tumor tissue treated with 25 mM D-allose for 2 or 3 weeks. For the Western blot analyses, proteins were separated on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, blocked with 5% (w/v) non-fat dried milk in PBS, and incubated with anti-TXNIP (MBL, Nagoya, Japan), anti-TRX (MBL), and anti-GAPDH (14c10) antibodies (Cell Signaling Technology, Inc., Tokyo, Japan). Membranes were probed with a horseradish peroxidase-conjugated anti-mouse IgG (Amersham, Tokyo, Japan), and signals were detected using an enhanced chemiluminescence system (Amersham).
10
Quantification of Secreted BMP7 Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To allow analysis of BMP7 production following transient transfection in mammalian cells, conditioned media was concentrated 10-fold using Nanosep microconcentrators (10 kDa; Pall Life Sciences, Port Washington, NY). Concentrated conditioned media was combined with NuPAGE loading dye (Invitrogen, Carlsbad, CA) and nonreduced samples were loaded on 10% SDS-PAGE gels (Bio-Rad, Hercules, CA). Following electrophoresis, proteins were transferred onto ECL Hybond membranes (GE Health Care, Buckinghamshire, UK). Blots were blocked in 1% BSA in TBS-Tween buffer (Tris-buffered saline with 0.05% Tween-20) for a minimum of one hour, and then probed with a BMP7 antibody (MAB3542, R&D Systems, Minneapolis, MN) diluted in TBS-Tw buffer (1:5000) overnight. Bound primary antibody was detected with horseradish peroxidase–conjugated antimouse IgG (Amersham), diluted in TBS-Tw buffer (1:10,000). Western blots were developed using Lumi-light chemiluminescence substrates and detected on a Biorad Chemidoc instrument. The levels of mature BMP7 were quantified by densitometry, as a mean of n = 3 experiments.
Total protein content in the BMP7 conditioned media samples was determined using a PierceTM BCA protein assay (Thermo Scientific, Waltham, MA) according to the manufacturer’s guidelines.
Total protein content in the BMP7 conditioned media samples was determined using a PierceTM BCA protein assay (Thermo Scientific, Waltham, MA) according to the manufacturer’s guidelines.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!