To analyze ER stress marker proteins, total proteins collected from the transverse colons of No, Lop+ vehicle and Lop+AEtLP treated rats were separated by 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, after which the resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was subsequently incubated separately overnight at 4℃ with the following primary antibodies: eIF2α (Cell Signaling Technology Inc., Cambridge, MA, USA), p-eIF2α (Cell Signaling Technology Inc.), IRE1β (Santa Cruz Biotechnology, Santa Cruz, CA, USA), JNK (Cell Signaling Technology Inc.), p-JNK (Cell Signaling Technology Inc.) and anti-GAPDH (Cell Signaling Technology Inc.). Next, the membranes were washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.05% Tween 20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories, South San Francisco, CA, USA) at a dilution of 1:1,000 and room temperature for 2 h. Finally, the membrane blots were developed using Chemiluminescence Reagent Plus kits (Pfizer, New York, NY, USA and Pharmacia, New York, NY, USA).
Horseradish peroxidase conjugated goat anti rabbit igg
Manufactured by Zymo Research
Sourced in United States
Horseradish peroxidase-conjugated goat anti-rabbit IgG is a secondary antibody used in immunoassays and other immunological techniques. It consists of goat-derived antibodies specific to rabbit immunoglobulin G (IgG), which are conjugated to the enzyme horseradish peroxidase. This conjugate can be used to detect and quantify the presence of rabbit IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence reagent plus kits, by Pfizer (5 mentions)
Anti actin, by Merck Group (3 mentions)
Anti p pkc, by Cell Signaling Technology (3 mentions)
Anti pkc, by Cell Signaling Technology (3 mentions)
Eif2α, by Cell Signaling Technology (2 mentions)
Ire1β, by Santa Cruz Biotechnology (2 mentions)
P eif2α, by Cell Signaling Technology (2 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti p jnk, by Cell Signaling Technology (1 mentions)
Anti p53, by Abcam (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Anti bax, by Abcam (1 mentions)
Ecl western blot detection reagent, by Cytiva (1 mentions)
0.2 μm nitrocellulose membrane, by Bio-Rad (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ecl reagent, by Cytiva (1 mentions)
Mbs8242404, by MyBioSource (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
10 protocols using horseradish peroxidase conjugated goat anti rabbit igg
1
Analyzing ER Stress Markers in Colonic Tissues
2
Transverse Colon Protein Analysis
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Total proteins collected from the transverse colons of all subset groups (No, Lop+vehicle, Lop+GEGR) were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, after which the resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was then incubated separately with the following respective primary antibody, overnight at 4℃: anti-PKC (Cell Signaling Technology Inc.), anti-p-PKC (Cell Signaling Technology Inc.), anti-Bax (Abcam, Cambridge, UK), anti-p53 (Abcam, Cambridge, UK), anti-JNK (Cell Signaling Technology Inc.), anti-p-JNK (Cell Signaling Technology Inc.) or anti-actin (Sigma-Aldrich Co.). Next, the membranes were washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.05% Tween 20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories, South San Francisco, CA, USA) at a dilution of 1:1,000 for 2 h at room temperature. Finally, the membrane blots were developed using Chemiluminescence Reagent Plus kits (Pfizer, New York, NY, USA and Pharmacia, New York, NY, USA).
3
Western Blot Analysis of Protein Targets
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Proteins were separated on 10% polyacrylamide gels and transferred to 0.2 μm nitrocellulose membrane (Bio-Rad) in transfer buffer (25 mM Tris, 192 mM glycine and 10% methanol). Membranes were dried and blocked for 60 min at room temperature with TBST-1X (137 mM NaCl, 20 mM Tris, 0.1% Tween-20, at pH 7.6) containing 5% BSA (Sigma-Aldrich) and incubated overnight at 4 °C with rabbit anti-GAPDH (1:2000 Cell Signaling), rabbit anti-ANGPT1 (1:2000 Abcam), rabbit anti-CREB5 (1:1000 Abcam), and rabbit anti-TGFβR2 (1:1000 Abcam) antibodies. Membranes were washed three times in TBST-1X and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2,500, Zymed). Signal was detected and developed using ECL Western blot detection reagent (Amersham).
4
Quantifying Aortic eNOS Protein Levels
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Aortic tissue was homogenized and 6 × 106 HUVEC cells were sonicated in PBS buffer containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Supernatants were stored at -80 °C until analyzed. Western blot analysis was performed essentially as described in detail previously [8 (link)]. Rabbit polyclonal anti-eNOS (1:200 dilution, #MBS8242404, MyBiosource Inc., San Diego, CA, USA) and anti-actin (1:1000, #AANO1, Cytoskeleton Inc. Denver, CO, USA) for 1 h at room temperature. Horseradish peroxidase–conjugated goat anti-rabbit IgG (1:10,000 dilution; Zymed Laboratories, San Francisco, CA) was used as a secondary antibody, and immunoreactive proteins were visualized by chemiluminescence via ECL reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK). All Western blots were quantified by densitometric analysis using NIH ImageJ 1.44p software.
5
Immunoblotting Analysis of ER Stress Markers
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To investigate an alteration in the expression of ER stress marker proteins, total proteins collected from the transverse colons of No-, Lop+Vehicle and Lop+Urd treated rats were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, after which the resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was subsequently separately incubated overnight at 4℃, with the following primary antibodies: eIF2α (Cell Signaling Technology Inc., Cambridge, MA, USA), p-eIF2α (Cell Signaling Technology Inc.), IRE1β (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p- IRE1β (Santa Cruz Biotechnology) and anti-β-actin (Cell Signaling Technology Inc.). The membranes were then washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.05% Tween 20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories, South San Francisco, CA, USA) at a dilution of 1:1,000 at room temperature for 2 h. Finally, the membrane blots were developed using Chemiluminescence Reagent Plus kits (Pfizer, New York, NY, USA and Pharmacia, New York, NY, USA). The chemiluminescence signals that originated from specific bands were detected using FluorChemi®FC2 (Alpha Innotech Co., San Leandro, CA, USA).
6
INPP5E Protein Expression in Olfactory Mucosa
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Freshly dissected olfactory mucosa was homogenized on ice in a lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, and 1% Triton-X-100 complemented with protease inhibitors) for 20 min. The sample was centrifuged at 11,300 g for 10 min at 4°C. The supernatant was then used for a protein concentration assay, using the Bradford detergent-compatible assay according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Samples were heated for 2 min at 95°C in SDS loading buffer. A total of 30 μg of cell lysis samples were run on a 4–12% Bis-Tris acrylamide gel (Invitrogen). After electrophoretic transfer to nitrocellulose, membranes were incubated with 5% fat-free milk and then with the anti-INPP5E (Proteintech, 17797-1-AP) or anti-actin antibodies (A5060, Sigma) (diluted 1:500 and 1:1000, respectively). Bound primary polyclonal antibody was detected with a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-rabbit-IgG (Zymed). The Renaissance western blot chemiluminescence reagent was used according to the manufacturer's protocol (Perkin Elmer Life Sciences, Wellesley, MA). Images were captured using the EpiChemi3 Darkroom (UVP, Upland, CA). Intensity of specific bands were measured in NIH ImageJ and presented as a ratio between INPP5E and actin signal.
7
Western Blot Analysis of Colonic Proteins
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Total proteins (30 mg) collected from the transverse colons of subset groups (Vehicle, LoLop and HiLop treated ICR mice) were separated by 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, after which the resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was then incubated separately with primary antibody, anti-Gα (Abcame, Cambridge, UK), anti-PKC (Cell Signaling Technology Inc., Cambridge, MA, USA), anti-p-PKC (Cell Signaling Technology Inc.), or anti-actin (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4℃. Next, the membranes were washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.05% Tween 20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories, South San Francisco, CA, USA) at a dilution of 1:1,000 and room temperature for 2 h. Finally, the membrane blots were developed using Chemiluminescence Reagent Plus kits (Pfizer, New York, NY, USA and Pharmacia, New York, NY, USA).
8
Protein Expression Analysis in Rat Colon
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Total proteins collected from the transverse colons of subset groups (No, GEGR, Lop+vehicle, Lop+LoGEGR, Lop+MeGEGR, Lop+HiGEGR and Lop+BS treated SD rats) were separated by 4%–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, after which the resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was then incubated separately with primary antibody, anti-mAChR M2 antibody (Alomone Labs, Jerusalem, Israel), anti-PI-3K (Cell Signaling Technology Inc., Cambridge, MA, USA), anti-p-PI3K (Cell Signaling Technology Inc., Cambridge, MA, USA), anti-mAChR M3 antibody (Alomone Labs, Jerusalem, Israel), anti-PKC (Cell Signaling Technology Inc.), anti-p-PKC (Cell Signaling Technology Inc.), anti-Gα (Abcame, Cambridge, UK) or anti-actin (Sigma-Aldrich Co.) overnight at 4°C. Next, the membranes were washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.05% Tween 20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories, South San Francisco, CA, USA) at a dilution of 1:1,000 and room temperature for 2 h. Finally, the membrane blots were developed using Chemiluminescence Reagent Plus kits (Pfizer, New York, NY, USA and Pharmacia, New York, NY, USA).
9
Quantification of COX-2 and 4-HNE Proteins
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Protein concentrations in all samples were measured spectrophotometrically by a protein assay reagent kit (Pierce, Rockford, IL, USA) via a modified Bradford method. Bovine serum albumin was used as standard. Western blot analysis was performed as previously described [33] (link). A rabbit polyclonal COX-2 antibody (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a rabbit polyclonal 4-HNE antibody (1:1000 dilution) were used for Western blot (WB) analysis. Horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10000 dilution; Zymed Laboratories, San Francisco, CA, USA) was used as a secondary antibody and immunoreactive proteins were visualized on high performance chemiluminescence film via ECL (enhanced chemiluminescence) reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK). Western blotting was performed two times. Densitometric analysis of immunoblots was performed using NIH image 1.61 software for windows (Scion Corporation, Frederick, MD, USA).
10
Western Blot Analysis of Synaptic Proteins
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All subcellular fractions were subjected to 7.5% SDS-PAGE, and proteins were transferred to a PVDF membrane. After blocking for 1 h at room temperature with 3% skimmed milk in TBS-T buffer consisting of 0.1% Triton X-100, 150 mM NaCl, and 10 mM Tris-HCl (pH 7.5), the membrane was incubated at 4 °C overnight with rabbit anti-Caskin1/2C (1 μg/ml), anti-Caskin1 (1 μg/ml) and anti-PSD-95 (1:1000) antibodies. The membrane was then washed with the TBS-T buffer and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:20,000; Zymed) or goat anti-mouse IgG (1:20,000; GE Healthcare). It was then washed 4 times with the TBS-T buffer. The immunoreactivity was detected by use of an enhanced chemiluminescence detection kit (ECL, GE Healthcare, and Chemi-Lumi One Super, Nacalai Tesque).
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