All chemicals used in the present study were of either analytical or molecular biology grades and were obtained from commercial sources. Media components were purchased from Difco (USA). Oligonucleotides were purchased from Sigma (India) and IDT (USA). Restriction enzymes, Vent DNA polymerase, and other DNA-modifying enzymes were obtained from New England Biolabs (USA). Gel-extraction kits, plasmid miniprep columns, and the Ni-NTA agarose resin were obtained from Qiagen (Germany). Hybridization nitrocellulose membrane (filter type 0.45 μm) and Luminata forte Western horseradish peroxidase (HRP) substrate was obtained from Millipore (India). Anti-His mouse monoclonal antibody (27E8) and horse anti-mouse HRP-linked antibody were procured from Cell Signaling Technology (USA). Anti-GSH mouse monoclonal antibody (ab19534) was from Abcam (UK). Alexa Fluor 488–conjugated goat anti-mouse antibody was obtained from Molecular Probes (USA). Coelentrazine was purchased from Promega (USA).
Luminata forte western horseradish peroxidase hrp substrate
Manufactured by Merck Group
Sourced in India
Luminata Forte Western horseradish peroxidase (HRP) substrate is a reagent used in Western blot analysis. It is a chemiluminescent substrate that reacts with HRP-conjugated secondary antibodies to produce a light signal, which can be detected and quantified to measure the abundance of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (2 mentions)
Gapdh, by Merck Group (2 mentions)
Oligonucleotide, by Merck Group (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Ni nta agarose resin, by Qiagen (1 mentions)
Alexa fluor 488 conjugated goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Plasmid miniprep columns, by Qiagen (1 mentions)
Coelentrazine, by Promega (1 mentions)
Horse anti mouse hrp linked antibody, by Cell Signaling Technology (1 mentions)
Vent dna polymerase, by New England Biolabs (1 mentions)
Bolt bis tris plus, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Bio-Rad (1 mentions)
Rabbit anti pparγ, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated anti mouse secondary antibody, by Agilent Technologies (1 mentions)
Tyrosinated tubulin, by Merck Group (1 mentions)
Acetylated tubulin, by Merck Group (1 mentions)
Bolt 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Complete mini protease inhibitor cocktail tablet, by Roche (1 mentions)
Phosphatase inhibitor cocktail, by AG Scientific (1 mentions)
3 protocols using luminata forte western horseradish peroxidase hrp substrate
1
Molecular Cloning and Protein Purification
2
Quantification of PPARγ Protein in Mouse Hippocampus
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Western blotting was performed as previously described [38] with modifications. Protein samples from the HP of all eight groups of mice (Table 2) were used. Proteins (15µg) were separated on 4-20% Bolt Bis-Tris Plus gels (Invitrogen), as described previously [15, 25] . The gel was subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) and blocked with 5% (w/v) non-fat milk tris-buffered saline (TBS) with 0.1% Tween-20 (Bio-Rad) for 1 hour at room temperature. The membrane was then incubated with rabbit anti-PPARγ (1:1000, Cell Signaling) primary antibody at 4°C overnight. Subsequently, a corresponding anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000; DAKO) was applied. Binding was visualized with enhanced chemiluminescence solution using Luminata Forte Western horseradish peroxidase (HRP) substrate (Millipore). Band intensity was measured as the sum optical density by using ImageJ software (version1.4; NIH) and normalized to control labelling of glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000, Millipore).
3
Axon Protein Extraction and Quantification
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Plating media was removed from microfluidic chambers and culture was rinsed with HBSS. Axons were harvested with RIPA buffer with protease (CompleteTM Mini Protease Inhibitor Cocktail tablets, Roche) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail, A.G. Scientific), with protein pooled from 2 to 4 chambers per treatment group. Denatured protein samples (20 μg) were electrophoresed into Bolt®; 4–12% Bis-Tris gels (Invitrogen), transferred to PVDF membrane (Bio-Rad) and incubated overnight with primary antibodies acetylated tubulin (1:1,000 mouse, Sigma), tyrosinated tubulin (1:1,000 rabbit, Millipore), MAP1B (1:1,000 mouse, AbCam). The corresponding anti-rabbit or anti-mouse horseradish peroxidase conjugated secondary antibody (1:7,000, DAKO) was used as previously described (King et al., 2018 (link)). GAPDH (1:5,000 mouse, Millipore) was used as a loading control. Bands were visualized with enhanced chemiluminescence (ECL) solution-Luminata Forte Western horseradish peroxidase (HRP) substrate (Millipore) and images acquired with a Chemi-Smart 5000 Imaging System (Vilber Lourmat) equipped with Chemi-Capt 5000 software. Band intensity was measured as the integrated intensity using Fiji software. After standardizing to GAPDH, each value was calculated as a percentage of control samples (See Supplementary Figure 1 ).
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