Expression of GRP78, CHOP, caspase-12, total and phospho-(Ser473)-Akt (p-Akt) were measured by western blot. Left ventricular tissues were homogenized using lysis buffer (Beyotime, China), and the supernatants were collected after centrifugation at 12,000 × g for 15 min at 4°C. After quantitative analysis of protein concentration, total proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), blocked with 5% non-fat milk in Tris buffered saline for 1 h at 37°C, and then incubated overnight at 4°C with anti-Akt (Cell signaling, dilution: 1:1000), anti-phospho-Akt (Ser473, Santa Cruz, dilution: 1:200), anti-caspase-12 (Santa Cruz, dilution: 1:200), anti-GRP 78 (Signalway Antibody Co., Ltd, dilution: 1:3000), and anti-CHOP (Beyotime, dilution: 1:1000) as primary antibodies. After incubation for 1 h at 37°C with secondary antibody, bands were seen using the enhanced chemiluminescence kit (Beyotime) according to the manufacturer's protocol. All the results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels and expressed as fold intensity compared with the Sham group.
Anti caspase 12
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-caspase-12 is a laboratory reagent used for the detection and quantification of caspase-12, a key enzyme involved in the apoptosis (programmed cell death) pathway. This product provides a reliable tool for researchers to study the role of caspase-12 in various cellular and biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti grp78, by Santa Cruz Biotechnology (2 mentions)
Anti phospho akt ser473, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence kit, by Beyotime (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti chop, by Beyotime (1 mentions)
X omat film, by Kodak (1 mentions)
Anti parp, by Santa Cruz Biotechnology (1 mentions)
Anti phospho perk, by Santa Cruz Biotechnology (1 mentions)
Anti caspase 7, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti chop, by Santa Cruz Biotechnology (1 mentions)
Anti grp94, by Santa Cruz Biotechnology (1 mentions)
Anti eif2α, by Cell Signaling Technology (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
5 protocols using anti caspase 12
1
Evaluating Apoptosis Markers in Cardiac Tissue
2
Protein Expression Analysis by Western Blotting
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Whole cell lysates were prepared and analyzed by Western blotting as described previously [32 (link)]. Proteins in cell lysates were separated by precast 8-20% SDS-polyacrylamide gel electrophoresis, and then electrophoretically transferred from the gel onto polyvinylidene difluoride membranes. After blocking, blots were incubated with anti-GRP78, anti-GRP94, anti-phospho-PERK, anti-caspase-12, anti-caspase-7, anti-CHOP, anti-PARP, and anti-β-actin antibodies (Santa Cruz Biotechnology) and anti-eIF2α (Cell Signaling Technology, Danvers, MA, USA) in PBS within 0.1% Tween 20 for 1 h followed by two 15 min washes in PBS with 0.1% Tween 20. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 60 min. Detection was performed with Western blotting reagent ECL (Amersham-GE Healthcare Life Sciences, Pittsburgh, PA, USA), and chemiluminescence was exposed by the Kodak X-Omat films.
3
Western Blot Analysis of Cellular Proteins
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Total proteins were electrophoretically separated on sodium dodecyl sulfate polyacrylamide gels (10-12%) according to the molecular size of the target protein, and were subsequently transferred onto polyvinylidene difluoride membranes. After being blocked with 5% skim milk, the membranes were incubated at 4° C overnight with the following primary antibodies: anti-CCDC170 (1:500, PA5-34723, Thermo Fisher Scientific, USA), anti-XBP1 (1:1000, PA5-27650, Thermo Fisher Scientific, USA), anti-IRE1α (1:1000, 14C10, Cell Signaling Technology, USA), anti-cleaved PARP (1:1000, 9542S, Cell Signaling Technology), anti-Caspase-12 (1:500, sc-21747, Santa Cruz Biotechnology, USA), anti-Caspase-7 (1:1000, D2Q3L, Cell Signaling Technology, USA), anti-Bcl2 (1:500, ab692, Abcam, USA) and anti-β-actin (1:2000, Santa Cruz Biotechnology, USA). Then, the membranes were washed thoroughly and incubated with secondary antibodies (1:3000 anti-mouse or 1:5000 anti-rabbit) at room temperature for 1 hour. The signals were visualized using the enhanced chemiluminescence method (Immobilon Western Chemiluminescent HRP Substrate, Millipore, USA). The samples were analyzed in duplicate, and the experiment was performed three times.
4
Western Blotting of ER Stress Proteins
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Cell pellets were prepared as described53 (link) and western blotted with the following antibodies: anti-IRE1α (ab37073; 1 : 1500) from Abcam (Cambridge, MA, USA); anti-β-actin (A1978; 1 : 5000) from Sigma-Aldrich; anti-PERK (sc-13073; 1 : 1000), anti-ATF6α (sc-22799; 1 : 1000), anti-GADD153 (sc-575; 1 : 1000), anti-ASK1 (sc-7931 and sc-5294; 1 : 1000 for WB, 1 : 100 for IP and PLA), anti-caspase-12 (sc-70227; 1 : 1000), anti-GRP78 (sc-1050; 1 : 1000), anti-TRAF2 (sc-7346; 1 : 1000), and anti-calpain (sc-7530; 1 : 1000) (Santa Cruz Biotechnology); anti-ryanodine receptor (MA3-916; 1 : 1000) (Thermo Scientific, Hudson, NH, USA); anti-InsP3R (07-1210; 1 : 2000 for WB, 1 : 300 for IP, 1 : 100 for PLA) and anti-CIB1 (MAB2601; 1 : 1500 for WB, 1 : 500 for IP, 1 : 100 for PLA) (Millipore, Schwalbach, Germany); and anti-calreticulin (2891; 1 : 2000), anti-calnexin (2433; 1 : 2000), anti-cleaved caspase-9 (9501; 1 : 2000), anti-caspase-9 (9502; 1 : 2000), and anti-caspase-3 (9662; 1 : 2000) (Cell Signaling Technology, Beverly, MA, USA). Immunoreactive bands were photographed and quantified on LAS-3000 with MultiGauge (Fuji Film Inc., Tokyo, Japan).
5
Antibody Detection in Inflammatory Response
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Aldo, 2',7'-dichlorofluorescein diacetate, and MT were purchased from Sigma (St. Louis, MO). Mouse monoclonal anti-Nlrp3 antibody and rabbit polyclonal anti-ASC antibody were purchased from adipoGen (San Diego, CA). Rabbit polyclonal anti-IL-18 and anti-caspase-12 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-GRP78, anti-GRP94, and anti-CHOP were purchased from Cell Signaling Technology. Goat polyclonal anti-IL-1b was purchased from R&D systems (Minneapolis, MN).
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