Cell extracts were diluted in sample buffer (50 mmol/L Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 6% 2-mercaptoethanol, and 0.0025% bromophenol blue) and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples were loaded at the same protein concentration for each experiment. The primary antibodies used were anti-LMP1 antibody (S12) at 1:50, anti-actin antibody (AC-74, Sigma, St. Louis, MO) at 1:5000, anti-phospho-AKT antibody (#4058, Cell Signaling Technology, Danvers, MA) at 1:1000, anti-AKT antibody (#9272, Cell Signaling Technology) at 1:1000, anti-NFκB (p65) antibody (610868, BD Biosciences, Franklin Lakes, NJ) at 1:250, anti-IκBα antibody (#4814, Cell Signaling Technology) at 1:1000, anti-caspase-3 antibody (#9662, Cell Signaling Technology) at 1:1000, and anti-poly(ADP-ribose) polymerase (PARP) antibody (C-2-10, Sigma) at 1:2000. The secondary antibodies used were Goat Anti-Mouse Ig's HRP Conjugate (AMI3404, BioSource International, Camarillo, CA) and HRP-Goat Anti-Rabbit IgG (H+L) (656120, Invitrogen, Carlsbad, CA). The bands were visualized using WEST-oneTM Western Blot Detection System (iNtRON Biotechnology, Seongnam, Korea) or Chemi-Lumi One Super (Nacalai tesque, Kyoto, Japan).
C21 0
Manufactured by Merck Group
C21:0 is a long-chain fatty acid that can be used as a reference standard or an analytical tool in various laboratory applications. It is a saturated fatty acid with 21 carbon atoms.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg h l hrp, by Thermo Fisher Scientific (1 mentions)
Anti actin antibody ac 74, by Merck Group (1 mentions)
Anti phospho akt antibody, by Cell Signaling Technology (1 mentions)
Anti caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Anti iκbα antibody, by Cell Signaling Technology (1 mentions)
Anti akt antibody, by Cell Signaling Technology (1 mentions)
West one western blot detection system, by iNtRON Biotechnology (1 mentions)
Chemi lumi one super, by Nacalai Tesque (1 mentions)
Cp 3800, by Agilent Technologies (1 mentions)
Cp 3800 gas chromatograph, by Agilent Technologies (1 mentions)
Wax 10, by Merck Group (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Api 4000 triple quadrupole mass spectrometer, by AB Sciex (1 mentions)
Hplc system, by Agilent Technologies (1 mentions)
Heneicosanoic acid, by Merck Group (1 mentions)
13c labeled linoleic acid, by Merck Group (1 mentions)
5 protocols using c21 0
1
Western Blot Analysis of Cellular Proteins
2
Fatty Acid Profiling in Feed, Grass, and Meat
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fatty acid intake was evaluated from the lipid fraction extracted from feed, grass and meat following the method reported by Folch et al. [24 (link)]. To measure the fatty acid methyl esters, the lipid extract was dried with a rotavapor, and 1 mL of n-exane was added. Finally, the trans-metylation procedure was performed with 0.5 mL of 2 M KOH methanol solution at 60°C for 15 min. One microliter was added to the gas chromatography system (CP 3800 VARIAN, Milan, Italy) equipped with an FID detector and a capillary column of 100 m length x 0.25 mm x 0.2 μm film (Supelco, Bellefonte, PA). To calculate the amount of each fatty acid, heneicosanoic acid was used as the internal standard (C21:0, Sigma–Aldrich analytical standard). The amount of each fatty acid was expressed as mg/100g of tissue and used to calculate the total saturated (SFA), total monounsaturated fatty acids (MUFA), and total PUFA from the n-3 and n-6 series.
3
Quantitative Lipid Profiling of Meat
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total lipids were extracted and quantified from the muscle (10 g), accurately minced by an ultraturrax (IKA T18, Steroglass, Italy) following the method reported by Folch et al.49 (link) To obtain fatty acid methyl esters, the lipid extract was dried with a rotary evaporator (Strike 10 Steroglass, Italy), and 1 ml of n-hexane was added. Finally, the trans-methylation procedure was performed with 0.5 ml of 2 M KOH–methanol solution at 60 °C for 15 min. To calculate the amount of each FA, heneicosanoic acid was used as the internal standard (C21:0, Sigma-Aldrich analytical standard), and data were expressed as mg/100 g of meat (quantitative evaluation) and % of total FA (qualitative evaluation). The average amount of each FA was used to calculate the sum of total PUFA and LC PUFA of the n-3 and n-6 series and the proportion of each LC PUFA respect to the total LC PUFA. Also the muscle enzyme activity was estimated as products/precursors ratio for both series.
4
Fatty Acid Profiling of Lipid Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lipids were extracted from the feed and different tissues according to Mattioli et al.29 (link). To obtain fatty acid methyl esters, the lipid extract was dried with a rotary evaporator (Strike 10 Steroglass, Italy), and 1 ml of n-hexane was added. Finally, the trans-methylation procedure was performed with 0.5 ml of 2 M KOH–methanol solution at 60 °C for 15 min. To calculate the amount of each FA, heneicosanoic acid was used as the internal standard (C21:0, Sigma-Aldrich analytical standard). The recovery rate of the internal standard in the testis was 83% ± 3%.
The FA composition was determined using a Varian gas chromatograph (CP-3800) equipped with a flame ionization detector and a capillary column 100 m long × 0.25 mm × 0.2 μm film (WAX-10; Supelco, Bellefonte, PA, USA). Helium was used as the carrier gas with a flow of 0.6 mL/min. The split ratio was 1 : 20. The oven temperature was programmed as reported by Mattioli et al.29 (link). Individual FA methyl esters (FAME) were identified by comparing the relative retention times of peaks in the sample with those of a standard mixture (FAME Mix Supelco, Sigma-Aldrich).
The FA composition was determined using a Varian gas chromatograph (CP-3800) equipped with a flame ionization detector and a capillary column 100 m long × 0.25 mm × 0.2 μm film (WAX-10; Supelco, Bellefonte, PA, USA). Helium was used as the carrier gas with a flow of 0.6 mL/min. The split ratio was 1 : 20. The oven temperature was programmed as reported by Mattioli et al.29 (link). Individual FA methyl esters (FAME) were identified by comparing the relative retention times of peaks in the sample with those of a standard mixture (FAME Mix Supelco, Sigma-Aldrich).
5
Quantification of Fatty Acids in Obese Mouse Livers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Livers from HFD fed obese mice treated with vehicle or with MS-275 were homogenized in methanol with tissue lyser (Qiagen). For quantitative analysis of fatty acids, aliquots of methanolic extracts after addition of internal standards (heneicosanoic acid, C21:0, and 13 C-labeled linoleic acid, Sigma-Aldrich) were subjected to acid hydrolysis and processed as previously described (26) . Fatty acid quantification was performed on an API-4000 triple quadrupole mass spectrometer (AB SCIEX) coupled with a HPLC system (Agilent) and CTC PAL HTS autosampler (PAL System).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!