The following antibodies were used: Goat anti-mouse amphiregulin (R&D Systems, catalogue number AF989; 1:1000), Mouse anti-nonphospho-Tyr1173-EGFR (Millipore, catalogue number 05-484; 1:1000), mouse anti-beta-actin (Santa Cruz, catalogue number sc-47778; 1:2000), rabbit anti-TACE/ADAM17 (Abcam, catalogue number ab39161; 1:2000), rabbit anti-HA (Santa-Cruz, catalogue number sc-805; 1:2000) and mouse anti-transferrin receptor (Invitrogen, catalogue number 13-6800; 1:1000). Corresponding species-specific HRP-coupled secondary antibodies were used from Santa Cruz and Cell Signaling.
Hrp coupled secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany
The HRP-coupled secondary antibody is a lab equipment product designed for use in various immunological techniques. It is a secondary antibody that has been conjugated with the enzyme horseradish peroxidase (HRP). This enzyme is used as a labeling agent to detect and amplify the signal from the primary antibody in assays such as ELISA, Western blotting, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Amersham hybond ecl, by GE Healthcare (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysate buffer, by Beyotime (2 mentions)
Dot blot system, by Bio-Rad (2 mentions)
Anti pts1 antibody, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Phosphoimager, by GE Healthcare (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Rabbit anti tace adam17, by Abcam (1 mentions)
Rabbit anti ha, by Santa Cruz Biotechnology (1 mentions)
Mouse anti transferrin receptor, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Sc 372, by Santa Cruz Biotechnology (1 mentions)
Nf κb p65 antibody, by Santa Cruz Biotechnology (1 mentions)
Lamin b antibody, by Santa Cruz Biotechnology (1 mentions)
Readyprep protein extraction kit cytoplasmic nuclear, by Bio-Rad (1 mentions)
Sc 6216, by Santa Cruz Biotechnology (1 mentions)
20 protocols using hrp coupled secondary antibody
1
Immunoblotting Antibodies and Detection
2
Isolation and Analysis of Nuclear Proteins
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Nuclear proteins were isolated from iris and ciliary body by using ReadyPrepTM Protein Extraction Kit (Cytoplasmic/Nuclear, Bio-Rad, Hercules, CA, USA). Protein concentration was adjusted equally with protein assay kit (Bio-RAD, Hercules, CA, USA), then re-suspended in 5x sample loading buffer, heated for 5 min at 95°C and separated on 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred onto a nitrocellulose membrane (AmershamTM HybondTM-ECL, GE Healthcare, UK), blocked with 5% Bovine albumin BSA (A9418, Sigma-Aldrich, USA), and incubated with NF-κBp65 antibody (1∶300, sc-372, Santa Cruz Biotechnology, INC.) and Lamin B antibody (1∶500, sc-6216, Santa Cruz Biotechnology, INC.) at 4°C overnight. After washing with TBS-0.05% tween-20 (TBST), HRP-coupled secondary antibodies (1∶1000, Santa Cruz Biotechnology, INC.) were applied to the membrane for 1 hour at room temperature, followed by three washes with TBST. The immunoreactive bands were visualized with enhanced chemiluminescence reagents (GE Healthcare, UK) and images were captured by the Universal Hood II image system (Bio-Rad Laboratories, Segrate, Italy). Band intensities of NF-κBp65 were normalized with those of internal control (Lamin B) using NIH Image J software (version 1.47).
3
Analyzing Proteins by Western Blotting
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For standard protein analysis protein lysates were obtained by lysing the cells in NP-40 lysis buffer or 1× Laemmli buffer supplemented with 100 units of Benzonase Nuclease (Sigma). Proteins were separated by SDS-PAGE (8–15%) and blotted on a nitrocellulose membrane (GE Healthcare). For Ponceau staining the membrane was stained with Ponceau solution (Sigma) for 5 min at RT with gentle rocking. The Ponceau solution was removed through several washes with ddH20. After saturating free binding sites with 5% non-fat milk powder in TBS-T the membrane was incubated with suitable primary antibodies, anti-ZRF1 antibody 1:1000 (Novus Biologicals #NBP2-12802), anti-DDB2 antibody 1:500 (Santa Cruz sc-81246) or anti-XPC antibody 1:500 (Santa Cruz sc-74410)for 16 h at 4 °C under rotation. After three times 10 min washing with TBS-T the membrane was incubated with matching HRP-coupled secondary antibodies (anti-mouse sc-516102 or anti-rabbit sc-2357; Santa Cruz Biotechnology) 1:5000 diluted for 1 h at RT followed by another three washing steps. Signals were detected by chemiluminescence of HRP-coupled secondary antibodies (Santa Cruz Biotechnology) on a ChemiDoc Imaging System. Uncropped blots are provided in the Source Data file.
4
Western Blot Analysis of Protein Levels
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5x 106 cells were lysed in 200 μl NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 10 mM Tris-HCL pH 7.4, 1mM EDTA pH 8.0, 3% Glycerol) for 2 h on ice. 30 μg of total cell lysate were submitted to SDS-PAGE under reducing conditions. Immunoblotting was performed on polyvinylidene difluoride (PVDF) membranes. Western blots were probed with the following primary antibodies: rat α-EBNA2 (R3; IgG2A; E. Kremmer), rat α-CBF1 (RBP-J 7A11, E. Kremmer), rat α-GST (GST 6G9, IgG2A, E. Kremmer), mouse α-EBF (Santa Cruz Biotechnology, sc-137065), goat α-BATF (B-ATF H-19, Santa Cruz Biotechnology, sc-15280), rabbit αIRF4 (IRF4H-140), and-GAPDH (EMD Millipore MAB374). HRP-coupled secondary antibodies (Santa Cruz Biotechnology) and an ECL kit (GE Healthcare) were used for visualization. For subsequent quantification of protein levels, exposed films were scanned in transmission mode and protein band intensities were determined by densitometry using ImageJ software (http://rsbweb.nih.gov/ij/ ) [74 (link)].
5
Molecular Mechanisms of Inflammatory Signaling
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Colon segments were homogenized in phosphate buffer saline (PBS) and the supernatant was collected after centrifugation (4 °C, 12,000 g, 15 min). The cultured cells were lysed in lysis buffer containing protease and phosphatase inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) and the supernatant was collected. The protein (30 µg) in supernatant was separated by 10% SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked in 5% (w/v) skim milk for 2 h at room temperature and immunoblotted with primary antibodies (Cell Signaling Technology, Danvers, MA) against the mouse p-p65, p-IκBα and IκBα, as well as antibody against the human PXR (Abcam Company, Cambridge, MA), respectively. Blots were incubated with HRP-coupled secondary antibodies (Santa Cruz Biotechnology, Dallas, TX) and observed by enhanced chemiluminescence (ECL) detection reagents (Thermo Scientific, Waltham, MA). Protein expressions were analyzed by a GS-700 imaging densitometer (Bio-Rad, CA). β -actin (Santa Cruz) was used as an internal control.
6
Western Blot Analysis of TMPRSS2 and Histone 3
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For standard protein analysis, protein lysates were obtained by lysing the cells in NP-40 lysis buffer or crude 1 × laemmli buffer supplemented with Benzonase. Proteins were running on SDS-PAGEs (8–15%) and blotted on a nitrocellulose membrane (GE Healthcare). After saturating free binding sites with 5% non-fat milk powder in 1X TBS-T, the membrane was incubated with TMPRSS2 (Santa Cruz ref# sc-515727) and Histone 3 (abcam ref# ab1791) antibody overnight at 4 °C under rotation. After three times 10 min washing with 1 × TBS-T, membrane was incubated with matching HRP-coupled secondary antibodies (anti-mouse or anti-rabbit (Santa Cruz Biotechnology) for 1 h at RT followed by another three washing steps. Signals were detected by chemiluminescence of HRP-coupled secondary antibodies (Santa Cruz Biotechnology) on a Gel Doc (Biorad). Uncropped blots are provided in the Source Data file instructions.
7
Protein Detection via Western Blotting
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Equal amounts of protein in each sample were loaded onto an SDS gel and transferred to immobilon polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membrane was incubated with designed primary antibodies overnight at 4°C. The positive signals from HRP-coupled secondary antibodies (Santa Cruz Biotechnology; Santa Cruz, CA, USA) were visualized by enhanced chemiluminescence detection kit (Thermo Scientific, Rockford, IL, USA). The densitometric analysis of the band intensities was measured and normalized to the band intensities of GAPDH using the ImageJ softwere (NIH, Bethesda, MD, USA).
8
Ubiquitination and Proteasome Assay
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Cycloheximide was purchased from Cell Signaling Technology (Danvers, MA, USA). MG132 was purchased from Beyotime Biotechnology (Shanghai, China). Puromycin, doxycycline, G418 and EZview Red Anti-Flag M2 Affinity Gel was obtained from Sigma (Sigma-Aldrich). Protein A&G Agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Glutathione-sepharose 4B beads were from GE Healthcare (München, Germany). Anti-K48 antibody was purchased from Cell Signaling Technology detecting the endogenous levels of ubiquitination. Anti-Flag, anti-BAF155, anti-Myc, anti-GAPDH were purchased from Cell Signaling Technology. Anti-PSMA7, anti-PSMC3, anti-PSMC1, anti-PSMA1, anti-HBx were purchased from Abcam (Cambridge, UK). HRP-coupled secondary antibodies were obtained from Santa Cruz Biotechnology.
9
Protein Extraction and Western Blot Analysis
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Total proteins in tissues or cells were extracted with RIPA lysate buffer (Beyotime Inc., China) including benzosulfonyl fluoride (Beyotime Inc., China) and protease inhibitor cocktail (Beyotime Inc., China). SDS-PAGE was applied to isolate equal amounts of protein and then samples were transferred to PVDF membrane (Millipore Inc., USA). The membrane was incubated overnight with the primary antibody at 4°C and then incubated with HRP coupled secondary antibody (Santa Cruz Inc., China). Anti-GAPDH antibody (Santa Cruz Inc., China) was utilized as an internal control. Protein expression levels were measured using ECL substrate (Beyotime Inc., China).
10
Protein Expression Analysis of Cell Signaling
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Cells were harvested and washed twice with PBS (pH 7.4, 0.15 M), and total protein was extracted with RIPA buffer (Beyotime, Shanghai, China). Approximately 30 µg of total protein was subjected to SDS-PAGE and transferred to PVDF membranes, which were then blocked with 5% skim milk in TBST and incubated with primary antibody (1:1000) containing 5% BSA overnight at 4 °C. The membranes were washed three times with TBST and incubated with HRP-coupled secondary antibody (1:4000; Santa Cruz, Dallas, TX, USA) for 2 h at room temperature using enhanced chemiluminescence reagents (Chemicon International, USA) exposure and then re-probed-with-anti-β-actin-antibody (Santa Cruz, Dallas, TX, USA) at a dilution of 1:2000 to confirm the same protein loading. Detection of VASH2, Smad4, Smad2 + 3, Mmp2, Bcl-2, Caspase3.
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