For western blot analysis, 30 μg of protein was loaded on 4–20% Tris-glycine polyacrylamide gels. The gels were transferred to polyvinylidene difluoride membrane using the iBlot Dry Blotting System (Life Technologies, Grand Island, NY). The membranes were blocked and probed with primary antibodies according to the manufacturers suggested dilutions: VLCAD, LCAD, HADHA (Abcam, Cambridge, MA), and β-actin (Sigma-Aldrich, St. Louis, MO). Incubation was done with appropriate secondary antibodies. Image analyses were performed using an Odyssey Imager (LiCor, Lincoln, NE).
Vlcad
Manufactured by Abcam
Sourced in United Kingdom
VLCAD is a protein that catalyzes the initial step in the breakdown of long-chain fatty acids. It is a key enzyme in the mitochondrial beta-oxidation pathway. VLCAD is responsible for the oxidation of fatty acids with chain lengths of 14 to 20 carbons.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using vlcad
1
Western Blot Protein Analysis
2
Immunohistochemical Analysis of Metabolic Markers
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Immunohistochemistry (IHC) of both tumor and paired normal mucosal tissues was performed as previously described [33 (link)]. Primary antibodies against FAS, CPT1A, MCAD, LCAD, VLCAD, and HADHA were obtained from Abcam (Cambridge, UK). Histopathological differentiation and expression of these proteins were evaluated independently by two pathologists (PS Wu and HP Lin) and quantified as previously described [4 (link),33 (link)]. A composite score using the staining intensity (0, 1+, 2+, and 3+) and the percentage of the area showing reactivity was generated. Scores were averaged over replicate cores to obtain the final IHC score for each tumor. Representative examples of each score are presented in Figure 1 . HPV p16 expression was evaluated with a CINtec p16 Histology kit following the manufacturer’s instructions (Ventana, Tucson, AZ, USA). Anti-Ki-67 antibody was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Ki-67 expression was assessed based on percentage of positive nuclear staining in tumor regions.
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