Trp53+/+, Trp53+/−, and Trp53−/− MEFs were treated with 10Gy irradiation to activate the p53 pathway. Equal quantities of cells from each group were pelleted, lysed with RIPA buffer (Sigma-Aldrich; Cat#: R0278-50ML), and utilized for immunoblotting experiments. Protein samples were collected from lysed cell pellets. Protein concentrations were normalized via both cell number and BCA assay (Thermo Fisher; Cat#: 23227). Samples were run through 4%–15% gradient polyacrylamide gels (BioRad; Cat#: 4561083EDU) and transferred onto nitrocellulose membranes (Thermo Fisher; Cat#: 88018). We used the Rabbit anti TRP53 primary antibody (Cell Signaling; Cat#: 9282) for detection of TRP53, along with goat anti-rabbit HRP-linked secondary antibody (Cell Signaling; Cat#: 7074S). Rabbit primary antibody was used for detection of ACTB (β-actin) (ABCAm: Cat#: ab8227), along with goat anti-rabbit HRP-linked secondary antibody (Cell Signaling; Cat#: 7074S) (Figure S1C ).
Goat anti rabbit hrp linked secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Goat anti-rabbit HRP-linked secondary antibody is a laboratory reagent used in immunoassays and other immunochemical techniques. It functions to detect and amplify the signal of primary antibodies raised in rabbit. The antibody is conjugated with horseradish peroxidase (HRP), an enzyme that can be used to generate a colorimetric or chemiluminescent signal when exposed to appropriate substrates.
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Lab products found in correlation
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Rabbit anti trp53 primary antibody, by Cell Signaling Technology (2 mentions)
Rabbit anti β tubulin, by Cell Signaling Technology (1 mentions)
Westernbright ecl kit, by Advansta (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti grp78, by Cell Signaling Technology (1 mentions)
M per extraction buffer, by Thermo Fisher Scientific (1 mentions)
Anti grp94, by Cell Signaling Technology (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Gradient polyacrylamide gel, by Bio-Rad (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Mouse monoclonal anti polyhistidine antibody, by Merck Group (1 mentions)
Horse anti mouse igg hrp linked, by Cell Signaling Technology (1 mentions)
5 protocols using goat anti rabbit hrp linked secondary antibody
1
p53 Pathway Activation Immunoblotting
2
Analysis of p53 Activation in MEFs
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Trp53 +/+ , Trp53 +/-, and Trp53 -/-MEFs were treated with 10Gy irradiation to activate the p53 pathway. Equal quantities of cells from each group were pelleted, lysed with RIPA buffer, and utilized for immunoblotting experiments. Protein samples were collected from cell pellets lysed with RIPA buffer. Protein concentrations were normalized via both cell number and BCA assay. Samples were run through 4-15% gradient polyacrylamide gels (BioRad; Cat#: 4561083EDU) and transferred onto nitrocellulose membranes (Thermo Fisher; Cat#: 88018). We used the Rabbit anti TRP53 primary antibody (Cell Signaling; Cat#: 9282) for detection of TRP53, along with goat anti-rabbit HRP-linked secondary antibody (Cell Signaling; Cat#: 7074S). Rabbit primary antibody was used for detection of ACTB (B-actin) (Abcam: Cat#: ab8227), along with goat anti-rabbit HRP-linked secondary antibody (Cell Signaling; Cat#: 7074S) (Figure S1C).
3
Western Blot Analysis of Colon Tissue Proteins
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The radioimmunoprecipitation assay lysis buffer was applied to extract the total protein from about 50 mg of colon tissue. After that, the BCA protein assay kit was used to quantify it. WB was carried out as stated44 (link) with the following antibodies: rabbit anti-ZO-1 (1:1000, Cat #NBP1–85046; Novus Biologicals, CO, USA), rabbit anti-Claudin-1 (1:1000, Cat #NBP1–77036, Novus Biologicals), rabbit anti-FXR (1:1000, Cat #12295, Cell Signaling Technology, MA, USA), rabbit anti-TGR5 (1:1000, Cat #NBP2–23669, Novus Biologicals), rabbit anti-β-tubulin (1:1000, Cat #2146, Cell Signaling Technology), and goat anti-rabbit HRP-linked secondary antibody (1:3000, Cat #7074, Cell Signaling Technology). Using the Western BrightTM ECL kit (Advansta, CA, USA), the membranes were visualized.
4
Western Blot Analysis of Stress Proteins
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Cells were lysed in M‐PER extraction buffer (Thermo Fisher Scientific) in the presence of protease inhibitors (Thermo Fisher Scientific). Five μg (Seps1 and glucose regulated protein (GRP) 94) or 10 μg (GRP78 and heme oxygenase‐1(HO‐1)) of total protein was separated on 4–20% acrylamide gradient gels (NuSep), and transferred to PVDF membranes. After blocking, membranes were incubated overnight with anti‐Seps1 (an in‐house rabbit polyclonal antibody against the COOH terminus of Seps1 generated as described here (Gao et al. 2003 , 2004 , 2007 )), anti‐GRP94 (Cell Signaling, Danvers, USA; #2104), anti‐GRP78 (Cell Signaling; #3183), or anti‐HO‐1 (Assay Designs, Ann Arbor, USA; #SPA‐894) antibodies. This was followed by an incubation with a goat anti‐rabbit HRP‐linked secondary antibody (Cell Signaling; #7074). Bands were detected using ECL chemiluminescence (Invitrogen) and analyzed using Quantity One® software (BioRad, Gladsville, AUS). The gels were stained with SimplyBlue™ SafeStain (Invitrogen) to validate even loading and protein expression was normalized to the optical density of the total protein per lane.
5
Antibodies for Amyloid Oligomer Detection
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The following antibodies were obtained from commercial sources: Rabbit polyclonal anti-Oligomer A11 (Thermo Fisher Scientific, Cat#AHB0052, RRID: AB_2536236), Rabbit polyclonal anti-Amyloid fibrils OC (Millipore Sigma, Cat#AB2286, RRID: AB_1977024), Goat anti-Rabbit HRP linked secondary antibody (Cell Signaling Technology, Cat# 7074, RRID: AB_2099233), Mouse monoclonal Anti-poly Histidine antibody (Sigma-Aldrich, Cat# H1029, RRID: AB_260015), and Horse anti-Mouse-IgG HRP linked (Cell Signaling Technology, Cat# 7076, RRID: AB_330924).
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