Western blot analysis
Description
Western blot analysis of hippocampus samples from healthy subjects and AD patients.
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Steps to reproduce
Brain samples were homogenized (100 µL for 10 mg of tissue) in 20 mM Hepes pH 8.0, 1 mM EGTA, 0.4% NP-40, and added of complete protease- (11836153001, Roche) and phosphatase-inhibitor (5870, Cell Signaling) cocktails following the manufacturer’s instructions. Brain samples were homogenized using a pellet micro-pestle and then subjected to sonication (3 cycles, 20 s each). The lysates were centrifuged at 13000 g for 10 min at 4 °C and, after discarding the pellet, the concentration of total proteins in the supernatant was determined using the Bradford reagent (500-0205, BioRad). For each sample 40 µg of total proteins were subjected to SDS-PAGE and transferred on a PVDF membrane using the Mini Trans-Blot Cell system (BioRad). The membranes were developed using rabbit anti-PHGDH (HPA024031, Sigma, dilution 1:1000), anti-PSAT (abin2856767, Antibodies online, 1:1000) or anti-PSP (PA5-22003, Invitrogen, dilution 1:1000) antibodies. The membrane was blocked overnight at 4 °C with 4% dried milk in Tris-saline buffer pH 8.0 added of 0.1% Tween 20 and subsequently incubated with primary antibodies diluted in 2% dried milk in Tris-saline buffer pH 8.0 added of 0.05% Tween 20 for 2 h at room temperature. After extensive washing, the membrane was incubated for 1 h at room temperature with rabbit IgG (Alexa-Fluor Plus 800, 1:20000 dilution in Tris-saline buffer pH 8.0 added of 0.05% Tween 20). Western blots were analyzed by Li-cor ImageStudio software: the intensity signal of each sample was normalized by the GAPDH signal (detected using a mouse anti-GAPDH, 1:2000, MA5-15738 Invitrogen and a mouse IgG IRDye 680 1:5000). The content of each protein was calculated by the software based on the intensity of known amounts of recombinant proteins and was related to the g of total proteins loaded into the gel. Controls included the addition of a known amount of recombinant proteins to the samples. Each sample was analyzed at least three times (in three different SDS-PAGE runs). The results were analyzed using Prism (Graphpad Software Inc.). The D’Agostino and Pearson normality test was used to analyze the distribution of variables: since a non-normal distribution was observed, a non-parametric approach was used for all statistical analysis. Variation of protein levels between controls and AD patients was evaluated using the two-tailed Mann-Withney test. A p value <0.05 was considered as statistically significant.