The whole-cell lysates were harvested in the RIPA buffer supplemented with protease and phosphatase inhibitors (Thermo Scientific). The protein concentration was determined using a BCA assay (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The cell lysates were separated using 10% SDS-PAGE gels, and the proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in PBST buffer for 1 h at room temperature and then incubated with the indicated primary antibodies overnight at 4 °C. After washing with PBST, the membranes were incubated with goat anti-rabbit or anti-mouse secondary antibodies (1:5000, Thermo Scientific) for 2 h at room temperature. The enhanced chemiluminescence (ECL, Pierce) was then applied, and the protein bands were imaged using a ChemiDoc Imaging System (Bio-Rad), which was quantified using the Image Lab software v6.0 (Bio-Rad). The primary antibodies used in this study included rabbit anti-ELK1 (1:1000, Abcam), rabbit anti-total MTOR (1:1000, Cell Signaling, Danvers, MA, USA), rabbit anti-phosphor-MTOR (1:1000, Cell Signaling), rabbit anti-total S6K1 (1:1000, Cell Signaling), rabbit anti-phosphor-S6K1 (1:1000, Cell Signaling), and mouse anti-β-actin (1:1000, Santa Cruz Biotech, Santa Cruz, CA, USA).
Goat anti rabbit or anti mouse secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat anti-rabbit or anti-mouse secondary antibodies are used in various immunoassay techniques to detect the presence of rabbit or mouse primary antibodies. These secondary antibodies are conjugated with reporter molecules, such as fluorescent dyes or enzymes, to enable visualization or quantification of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Image lab software v 6, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Rabbit anti phosphor mtor, by Cell Signaling Technology (1 mentions)
Gbox chemi xt4 system, by Syngene (1 mentions)
Enhanced chemiluminescence hrp substrate, by Merck Group (1 mentions)
Acetyl coa carboxylase, by Cell Signaling Technology (1 mentions)
Genetools software, by Syngene (1 mentions)
Phosphorylated acc, by Cell Signaling Technology (1 mentions)
Phosphorylated ampkα, by Cell Signaling Technology (1 mentions)
Pparα, by Proteintech (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Mab374, by Merck Group (1 mentions)
Ab137647, by Abcam (1 mentions)
Ab92611, by Abcam (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Alexa 546, by Thermo Fisher Scientific (1 mentions)
Motorized injector, by Stoelting (1 mentions)
4 protocols using goat anti rabbit or anti mouse secondary antibody
1
Western Blot Analysis of mTOR Signaling
2
Immunoblot Analysis of AMPK Signaling
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Proteins were extracted using RIPA lysis buffer, then resolved by 10% denaturing SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated with specific primary antibodies at 4°C overnight. The antibodies against AMPKα, Phosphorylated-AMPKα, Acetyl-CoA Carboxylase (ACC), and Phosphorylated-ACC were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibodies against SIRT1 and SREBP-1c were obtained from Abcam (Cambridge, MA, USA) and antibodies against suppressor of variegation 39 homolog 2 (SUV39H2), fatty acid synthase (FASN), acyl-Coenzyme A oxidase (ACOX), and peroxisome proliferator activated receptor-α (PPAR-α) from Proteintech (Wuhan, China). Subsequently, the membranes were incubated with goat anti-rabbit or anti-mouse secondary antibodies (Thermo Scientific, Rockford, IL, USA) at room temperature for 1 h. Next, the enhanced chemiluminescence HRP substrate (Millipore) was added. The signal of protein bands was acquired by GBOX Chemi XT4 System and was finally quantified by Gene Tools software (Syngene, Cambridge, UK).
3
Western Blot Analysis of Protein Expression
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Cells were harvested by trypsinization, lysed in 1× sodium dodecyl sulfate lysis buffer, and denatured for 10 min at 100 °C. After immunoblotting, the membranes were blocked with 5% nonfat dry milk in TBS/0.1% Tween-20 and then incubated with the primary antibodies in 1% nonfat dry milk in TBS/0.1% Tween-20. Subsequently, the blots were incubated with goat anti-rabbit or anti-mouse secondary antibody (Invitrogen, Carlsbad, CA) and visualized with enhanced chemiluminescence (Invitrogen, Carlsbad, CA).
The immunoreagents used for Western blotting were rabbit polyclonal antibody against Gli1 (Abcam, ab92611, diluted 1:500) and rabbit polyclonal anti-FoxM1 (Abcam, ab137647, diluted 1:500). Mouse monoclonal antibody against CCNB1 was purchased from Cell Signaling Technology (CST, 4135, diluted 1:2000), and anti-β-actin antibody (Anbo, E0012, diluted 1:5000) or anti-GAPDH antibody (Millipore, MAB374, diluted 1:2000) was used as a loading control.
The immunoreagents used for Western blotting were rabbit polyclonal antibody against Gli1 (Abcam, ab92611, diluted 1:500) and rabbit polyclonal anti-FoxM1 (Abcam, ab137647, diluted 1:500). Mouse monoclonal antibody against CCNB1 was purchased from Cell Signaling Technology (CST, 4135, diluted 1:2000), and anti-β-actin antibody (Anbo, E0012, diluted 1:5000) or anti-GAPDH antibody (Millipore, MAB374, diluted 1:2000) was used as a loading control.
4
Evaluating Nigral Neuroinflammation in Rats
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Rats received unilateral intra-substantia nigra injection of saline or MG132 (9.5 ng) under general anesthesia (0.8–1.5% isoflurane in 30% oxygen with air). The stereotaxic coordinates for substantia nigra were laternal +2.0 mm, antero-posterior from the bregma point -5.3 mm and dorsi-ventral +7.8 mm [24 (link)]. The solution was injected into the substantia nigra with 10 μl Hamilton syringe coupled to a motorized injector (Stoelting, Wood Dale, IL) at a rate of 0.2 μl/min and the needle was left in situ for at least 5 min after injection. After surgery, the rats were kept in the same cages for recovery. Rats expressing infection signs were excluded from the study. The rats were sacrificed using overdose isoflurane at 24 h after MG-132 injection. For double immunofluorescent labelling studies, the substantia nigra sections were stained with two primary antibodies, mouse anti- TH (1:1000) and rabbit anti-HO-1 (1:500), then with Alexa 488 (excitation 495 nm, emission 519 nm) or Alexa 546 (excitation 556 nm, emission 573 nm)-conjugated goat anti-rabbit or anti-mouse secondary antibody (1:500; Invitrogen, CA, USA). DAPI (1 μg/ml) was used to stain nucleus and the fluorescent images were obtained using a confocal microscope (model SP5 LAS; Leica, Heidelberg, Germany).
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