The intestinal tissues collected from mice were lysed in tissue lysis buffer (Thermo, MA, USA) supplemented with a complete protease inhibitor cocktail (Roche, NJ, USA). The protein concentration of the extracts was determined using a bicinchoninic cid assay (BCA). The same amount of protein from different groups was subjected to electrophoretic analysis and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibody overnight at 4 °C, then treated with the horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 60 min. The expression of β-actin was applied as a control in the quantitative analysis. Results were visualized using an immobilon-enhanced chemiluminescence (ECL) Ultra Western HRP Substrate (Millipore, MA, USA). The expression of the protein was determined with Quantity One Image software v1.8.0 (Bio-Rad). The following antibodies were used: SIRT3 (#2627, 1:1000, Cell Signaling Technology, MA, USA), SIRT3 (ab246522, 1:1000, Abcam, Cambridge, England), Zo-1 (ab276131, 1:1000, Abcam), and Claudin (ab211737, 1:1000, Abcam). HRP-conjugated anti-mouse or anti-rabbit secondary antibodies were obtained from Invitrogen.
Hrp conjugated anti mouse or anti rabbit secondary antibodies
Manufactured by Thermo Fisher Scientific
Sourced in United States
HRP-conjugated anti-mouse or anti-rabbit secondary antibodies are laboratory reagents used for the detection and visualization of target proteins in Western blotting and immunohistochemistry applications. These antibodies are conjugated with the enzyme horseradish peroxidase (HRP), which catalyzes a colorimetric or chemiluminescent reaction for signal generation.
Automatically generated - may contain errors
Lab products found in correlation
Ecl ultra western hrp substrate, by Merck Group (1 mentions)
Sirt3, by Cell Signaling Technology (1 mentions)
Ab246522, by Abcam (1 mentions)
Tissue lysis buffer, by Thermo Fisher Scientific (1 mentions)
Ab211737, by Abcam (1 mentions)
Ab276131, by Abcam (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Sirt3, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ecl detection kit, by Thermo Fisher Scientific (1 mentions)
β actin antibody, by Merck Group (1 mentions)
2 protocols using hrp conjugated anti mouse or anti rabbit secondary antibodies
1
Western Blot Analysis of Intestinal Proteins
2
Protein Expression Analysis by Western Blot
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Cells were collected and lysed in urine buffer supplemented with 1% protease and 1% phosphorylation inhibitors (Roche Diagnostics, US). Equal amount of total protein (30μg) for each sample was loaded to gradient SDS-PAGE gel. After electrophoresis, protein was transferred onto polyvinylidene fluoride membranes (Millipore, US). The membranes were blocked in TBS with 0.1% Tween-20 and 5% non-fat dried milk for 1 hour at room temperature. After washed for three times, the membranes were probed with primary antibodies overnight at 4°C. The following antibodies were used in this study: ARID1A (1:500, Bethyl Laboratory); ATM, p-ATM, CHK1, p-CHK1 (Ser345) (1:500, Cell Signaling); PTEN, PI3K, AKT, p-AKT (Ser473) (1:500, Abcam, MA); H2AX, γH2AX antibodies (1:1000, Millipore) and β-actin antibody (1:2000, Sigma, US). On the next day, membranes were washed for three times, and incubated with HRP conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000, Invitrogen, US) for 1 hour. Target proteins were visualized using an ECL detection kit (Thermo Fisher Scientific, US).
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