At 48 h after treatment, cells were washed with cold PBS and subjected to a lysis buffer. Protein lysates were separated using 8% SDS-polyacrylamide gel electrophoresis, then electro-transferred onto nitrocellulose filter membranes. The membranes were blocked with a buffer containing 5% non-fat milk in PBS with 0.05% Tween-20 for 2 h and incubated with primary antibody (anti-c-Myc) overnight. Then membranes were incubated with peroxidase-conjugated secondary antibodies (Milli-pore, Darmstadt, Germany) and developed with an enhanced chemiluminescence detection kit (Pierce, Rockford, IL). β-actin was used as blank control.
Peroxidase conjugated secondary antibody
Manufactured by Merck Group
Sourced in United States, United Kingdom, China, Japan, France, Germany
The Peroxidase-conjugated secondary antibody is a laboratory reagent used in various immunoassay techniques. It consists of a secondary antibody labeled with the enzyme peroxidase. This enzymatic label can be used to detect and amplify the signal from a target primary antibody, allowing for sensitive and quantitative analysis of target molecules in a sample.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (8 mentions)
Ecl system, by GE Healthcare (7 mentions)
Anti β actin, by Merck Group (4 mentions)
Nitrocellulose membrane, by GE Healthcare (4 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Gapdh, by Santa Cruz Biotechnology (4 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Whatman, by Cytiva (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Image lab software, by Bio-Rad (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti gapdh antibody, by Santa Cruz Biotechnology (3 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Enhanced chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Anti phospho erk, by Cell Signaling Technology (2 mentions)
Anti flag, by Merck Group (2 mentions)
96 protocols using peroxidase conjugated secondary antibody
1
Western Blot Analysis of c-Myc
2
Quantification of Soluble CD90 in Biofluids
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Soluble CD90 in urine and plasma concentration was tested with Human CD90 Immunoassay Quantikine ELISA following the manufacturer’s instructions (Biomatik, Canada). Urinary sCD90 level was normalized to urine creatinine concentration, indicated as urinary sCD90/Cr. Log2 transformed urinary sCD90/Cr and plasma sCD90 were used for all analyses.
To assess the expression of CD90 in kidney, we performed immunohistochemistry. Briefly, paraffin-embedded renal sections were deparaffinized, rehydrated and heat mediated antigen retrieval was performed with citrate buffer (pH 6.2). Sections were blocked with 3% BSA buffer and stained with a monoclonal antibody against CD90 (Abcam, Cambridge, MA; 1:200 dilution). The stained sections were then incubated with peroxidase-conjugated secondary antibodies (ZSGB-BIO, China) and developed using DAB substrate (Millipore, Bedford, MA, USA). Image-Pro Plus was used to quantify the value of mean optical density.
To assess the expression of CD90 in kidney, we performed immunohistochemistry. Briefly, paraffin-embedded renal sections were deparaffinized, rehydrated and heat mediated antigen retrieval was performed with citrate buffer (pH 6.2). Sections were blocked with 3% BSA buffer and stained with a monoclonal antibody against CD90 (Abcam, Cambridge, MA; 1:200 dilution). The stained sections were then incubated with peroxidase-conjugated secondary antibodies (ZSGB-BIO, China) and developed using DAB substrate (Millipore, Bedford, MA, USA). Image-Pro Plus was used to quantify the value of mean optical density.
3
Western Blot Analysis of ADAM17
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Cells were lysed in 20 mM Tris-HCl pH 7.4, 137 mM NaCl, 2 mM EDTA pH 8.0, 10 % glycerol, 1 % NP-40, protease inhibitor cocktail (Roche, Penzberg, Germany), 1.3 mM sodium orthovanadate, 10 mM 1-10-phenanthroline, and 1 μM BB94. Cell lysates were quantified by using the Pierce BCA Protein Assay kit (Pierce, Rockford, IL, USA), and equal amounts of protein were separated on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. These were blocked with 5 % bovine serum albumin or skim milk in TBS-Tween 0.1 % and then incubated with primary antibodies, and bound antibodies were detected with the appropriate peroxidase-conjugated secondary antibodies (Amersham GE Healthcare, Piscataway, NJ, USA) by using the ECL detection system (Millipore). To detect ADAM17, the same amount of protein from cell lysates was concentrated with wheat germ agglutinin (WGA)-agarose beads (Vector Laboratories, Peterborough, UK) for 2 h at 4 °C. Proteins were eluted directly in SDS-polyacrylamide gel electrophoresis loading buffer.
Determination of proteins in the collected conditioned media was performed following the same protocol after concentrating 100× the samples by using centricons (Amicon Ultracel 3K; Millipore). Densitometric quantification was performed by using ImageJ software.
Determination of proteins in the collected conditioned media was performed following the same protocol after concentrating 100× the samples by using centricons (Amicon Ultracel 3K; Millipore). Densitometric quantification was performed by using ImageJ software.
4
Western Blot Analysis of Cell Signaling
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AGS cells (1x105) were seeded in 6-well plates and grown in F-12 for 24 hours prior to transfection. 72 hours after transfection, the cells were washed with cold PBS and subjected to a lysis buffer (35 mM Tris-Cl [pH 6.8], 20 g/L sodium dodecyl sulfate [SDS], 100 mM dithiothreitol). Protein lysates (20 μg each) were separated using 8% SDS-polyacrylamide gel electrophoresis, then electro-transferred onto nitrocellulose filter membranes. The membranes were blocked with a buffer containing 5% nonfat milk in PBS with 0.05% Tween-20 for 2 hours and incubated overnight with antibody at 4°C. After a second wash with PBS containing 0.05% Tween-20, the membranes were incubated with peroxidase-conjugated secondary antibodies (Millipore, Darmstadt, Germany) and developed with an enhanced chemiluminescence detection kit (Pierce, Rockford, IL). GAPDH was used as a loading control. Antibodies for CDK4, p-Rb, ERK, Akt, p-ERK, p-Akt, c-Met and GAPDH were from Cell Signaling Technology (Beverly, MA).
5
Western Blot Protocol for Protein Analysis
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Cells were washed twice with cold PBS. Next, cells were lysed in RIPA buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethyl sulfonyl fluoride, complete protease and phosphatase inhibitor cocktail (Roche) for 5 min, and were collected by scraping. Lysates were cleared by centrifuging at 12,000 r.p.m. for 10 min. Protein was quantified by Bradford Assay. Sample proteins were separated by SDS–PAGE gel electrophoresis. Next, proteins were transferred from gel to a nitrocellulose membrane (Millipore). After that, membranes were blocked in 5% BSA TBST for 30 min at room temperature (RT) and then incubated overnight with indicated primary antibodies at 4 °C. After that, membranes were washed three times and incubated with peroxidase-conjugated secondary antibodies (1:5,000, Millipore) and detected with enhanced chemiluminescence (Millipore) on X-ray films (Kodak). Uncropped scans of all the blots are shown in Supplementary Fig. 8 .
6
Western Blot Analysis of ZNF24
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At 48 hours after treatment, cells were washed with cold PBS and subjected to a lysis buffer. Protein lysates were separated using 8% SDS-polyacrylamide gel electrophoresis, then electro-transferred onto nitrocellulose filter membranes. The membranes were blocked with a buffer containing 5% non-fat milk in PBS with 0.05% Tween-20 for 2 hours and incubated with primary antibody (anti-ZNF24) overnight. Then membranes were incubated with peroxidase-conjugated secondary antibodies (Millipore, Darmstadt, Germany) and developed with an enhanced chemiluminescence detection kit (Pierce, Rockford, IL). β-actin was used as blank control.
7
Western Blot Analysis of NOTCH1
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At 48 h after treatment, cells were washed with cold PBS and subjected to a lysis buffer. Protein lysates were separated using 8% SDS-polyacrylamide gel electrophoresis, then electro-transferred onto nitrocellulose filter membranes. The membranes were blocked with a buffer containing 5% non-fat milk in PBS with 0.05% Tween-20 for 2 h and incubated with primary antibody (anti-NOTCH1) overnight. Then the membranes were incubated with peroxidase-conjugated secondary antibodies (Milli-pore, Darmstadt, Germany) and developed using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL). β-actin was used as blank control.
8
Protein Extraction and Western Blot Analysis
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Proteins were extracted from PC cell lines using lysis buffer (Triton X-100, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA and 10% glycerol) containing a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) and phosphatase inhibitors (5 mM NaF, 200 µM Na3VO4 and 100 µM PMSF) and quantified by Bradford assay. Thirty micrograms of protein were separated with precast 4–15% Tris-Glycine SDS-polyacrylamide gels (MINI-PROTEAN TGX Gels, BioRad Laboratories, Hercules, CA, USA) and transferred onto PVDF membranes with the Trans-Blot Turbo Transfer System (BioRad). Membranes were blocked for 1 h in TBS-Tween supplemented with 5% of milk then probed with primary antibodies overnight at 4 °C. Membranes were washed with TBS-Tween three times for 10 min and probed with peroxidase-conjugated secondary antibodies (Millipore, Temecula, CA, USA) for 1 h. Chemiluminescence was enhanced by ECL prime (Amersham, GE Healthcare, Chicago, IL, USA) and detected using the ChemiDoc MP Imaging System (Bio-Rad). The antibodies used in this study were anti-IKKε (1/1000, Imgenex, San Diego, CA, USA), anti-Rad51 (1/1000, Abcam) and anti-ß-actin (1/2000, Abcam).
9
Protein Separation and Immunoblotting
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Proteins were separated on SDS 5–15% polyacrylamide gels (BIORAD, Hercules, CA) and either stained with Imperial protein stain (Pierce, Rockford, IL) or transferred to 0.45 μm pure nitrocellulose transfer and immobilization membrane (Schleicher and Schuell, Keene, NH). Immunoblotting employed peroxidase conjugated secondary antibodies (Chemicon International, Temecula, CA) and Supersignal West Pico chemiluminescent substrate (Pierce). The anti-actin C4 monoclonal antibody was purchased from Chemicon International. The anti-actin polyclonal antibody was purchased from Cytoskeleton, Inc. The anti-phosphotyrosine 4G10 monoclonal antibody was purchased from Upstate (Millipore). The anti-chlamydial EB polyclonal antibody, the Momp monoclonal antibody and the GAPDH monoclonal antibody were all purchased from Pierce. The anti-c-myc monoclonal antibody was purchased from Genscript (Piscataway, NJ). The anti-chlamydial Hsp60 A57-B9 monoclonal antibody was purchased from Thermo Fisher Scientific (Waltham, MA). Polyclonal rabbit antibodies directed toward C. trachomatis L2 LGV 434 Tarp (CT456) were developed at Rocky Mountain Laboratories as previously described (Clifton et al., 2004 (link)).
10
Proteomic Profiling of Dystrophin Complex
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For the gel-and liquid chromatography-based proteomic and biochemical profiling of the dystrophin complex and membrane fractions from skeletal muscles, analytical grade reagents and materials were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK), Sigma Chemical Company (Dorset, UK), Bio-Rad Laboratories (Hemel-Hempstead, Hertfordshire, UK) and National Diagnostics (Atlanta, GA, USA). Proteolytic digestion was carried out with sequencing grade modified trypsin and Lys-C enzymes from Promega (Madison, WI, USA). Materials for immunoblotting included Whatman nitrocellulose transfer membranes from Invitrogen (Carlsbad, CA, USA), chemiluminescence substrate from Roche Diagnostics (Mannheim, Germany) and the reversible membrane stain MemCode from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies were purchased from Abcam, Cambridge, UK (ab124798 to desmoglein isoform DSG1, ab2413 to fibronectin, ab58562 to biglycan, ab52488 to lactate dehydrogenase, ab14739 to the voltage-dependent anion-selective channel protein VDAC-1, and ab16048 to lamin-B1), and Sigma-Aldrich, Dorset, UK (L9393 to laminin). Peroxidase-conjugated secondary antibodies were obtained from Chemicon International (Temecula, CA, USA).
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