Cells were collected and lysed on ice. After centrifugation, protein concentration was measured using a BCA protein assay kit (ThermoScientific, USA). Equivalent amounts of protein (2–25 μg) onto 10–15% SDS-polyacrylamide gels. Then, protein was transferred onto a PVDF membrane and blocked with 5% bovine serum albumin solution for 1 h at room temperature. The membranes were then probed with primary antibodies (1 : 1000 dilution in 5% BSA in TBS) overnight at 4°C. Following the incubation, the membranes were washed using TBST and incubated with HRP (Horseradish peroxidase)-conjugated secondary antibodies (Santa Cruz Biotechnology, USA) for 1 h at room temperature. After washing, protein bands were detected with an enhanced chemiluminescence detection kit (Pierce, Rockford, lL, USA). Images were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA).
Hrp horseradish peroxidase conjugated secondary antibodies
Manufactured by Santa Cruz Biotechnology
Sourced in United States
HRP (horseradish peroxidase)-conjugated secondary antibodies are affinity-purified antibodies that have been labeled with the enzyme horseradish peroxidase. They are designed for use in various immunodetection techniques, such as Western blotting, ELISA, and immunohistochemistry, where they can bind to and signal the presence of primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (3 mentions)
Nc membrane, by Pall Corporation (3 mentions)
Phosphatase inhibitor cocktail, by Roche (2 mentions)
Protease inhibitors cocktail, by Roche (2 mentions)
Mouse anti goat, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
Anxa1, by Proteintech (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
5 protocols using hrp horseradish peroxidase conjugated secondary antibodies
1
Western Blot Analysis of Protein Expression
2
Western Blot Protocol for Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells or tissues were lysed by RIPA buffer (Thermo Fisher Scientific, 89,901) with protease inhibitors cocktail (Roche Diagnostics, 05892970001) and phosphatase inhibitor cocktail (Roche Diagnostics, 04906845001). The lysates were clarified by centrifugation at 13000g for 30 min at 4 °C. Protein concentrations were determined by BCA protein assay kit (Thermo Fisher Scientific, 23,225) followed by boiled with loading buffer. Protein samples (50–150 μg) were separated through SDS-PAGE, then transferred to NC membranes (Pall Corporation) blocked and incubated with the primary antibodies. After washing with TBST, the blots were incubated with goat anti-rabbit (Santa Cruz, sc-2004), goat anti-mouse (Santa Cruz, sc-2005) or mouse anti-goat (Santa Cruz, sc-2354) HRP (horseradish peroxidase)-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, 34,076).
3
Western Blot Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in cell lysis buffer containing protease inhibitor cocktail (Roche, Basel, Switzerland). The lysates were obtained by centrifugation at 13,000 rpm for 30 min at 4 °C. The concentration of total protein was calculated using a BCA protein assay kit (CW0014, CWBIO, China). Protein samples (50 μg) were loaded and the separated using 10% SDS-PAGE, transferred onto PVDF membranes (K5MA6041H, Millipore, USA), blocked with 5% skim milk-PBS solution, and probed with the primary antibodies. After washing with 0.1% Tween 20-PBS solution, the blots were incubated with goat anti-rabbit (Santa Cruz, sc-2004) or goat anti-mouse (Santa Cruz, sc-2005) HRP (horseradish peroxidase)-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, 34076).
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells or tissues were lysed with RIPA buffer (Thermo Fisher Scientific, 89901) containing protease inhibitors cocktail (Roche Diagnostics, 05892970001) and phosphatase inhibitor cocktail (Roche Diagnostics, 04906845001). The lysates were clarified by centrifugation at 13,000 g for 30 min at 4 °C. The total protein concentration was estimated using a BCA protein assay kit (Thermo Fisher Scientific, 23225). Protein samples (50–150 μg) were loaded on to and separated using SDS/PAGE, transferred on to NC membranes (Pall Corporation) blocked and probed with the primary antibodies. After washing, the blots were incubated with goat anti-rabbit (Santa Cruz, sc-2004) or goat anti-mouse (Santa Cruz, sc-2005) HRP (horseradish peroxidase)-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, 34076). The uncropped versions of western blots are shown in Supplementary Fig. 17 .
5
Western Blotting Procedure for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed according to the standard protocols. The total cellular or tissue protein was lysed by RIPA buffer supplemented with protease inhibitor and phosphatase inhibitor cocktail.
Protein content was quanti ed by BCA Protein Assay Kit (Thermo Fisher Scienti c, West Palm Beach, FL). Equal amounts (50 μg) of proteins were electrophoresed by 10% or 15% SDS polyacrylamide gels and were then transferred to NC membranes (Pall Corporation). The membranes were blocked with 5% skim milk for one hour and then incubated with the indicated primary antibodies at 4 °C overnight. After washing with TBST, the blots were labeled with HRP (Horseradish peroxidase)-conjugated secondary antibodies (Santa Cruz Biotechnology), and then detected using ECL kit (Pierce Biotech, Rockford, IL). The band intensities of the western blots were analyzed by ImageJ software. The antibodies used in this study were ANXA1 (#21990, 1:2000, Proteintech, China), LC3B (#2775, 1:1000, Cell Signaling Technology, USA), SQSTM1 (#18420, 1:2000, Proteintech, China). Tubulin (#SC8035, 1:1000, Santa Cruz, USA), p-AMPK (#2535, 1:1000, Cell Signaling Technology, USA), AKT (#4685, 1:1000, Cell Signaling Technology, USA), p-AKT (#4060, 1:2000, Cell Signaling Technology, USA).
Protein content was quanti ed by BCA Protein Assay Kit (Thermo Fisher Scienti c, West Palm Beach, FL). Equal amounts (50 μg) of proteins were electrophoresed by 10% or 15% SDS polyacrylamide gels and were then transferred to NC membranes (Pall Corporation). The membranes were blocked with 5% skim milk for one hour and then incubated with the indicated primary antibodies at 4 °C overnight. After washing with TBST, the blots were labeled with HRP (Horseradish peroxidase)-conjugated secondary antibodies (Santa Cruz Biotechnology), and then detected using ECL kit (Pierce Biotech, Rockford, IL). The band intensities of the western blots were analyzed by ImageJ software. The antibodies used in this study were ANXA1 (#21990, 1:2000, Proteintech, China), LC3B (#2775, 1:1000, Cell Signaling Technology, USA), SQSTM1 (#18420, 1:2000, Proteintech, China). Tubulin (#SC8035, 1:1000, Santa Cruz, USA), p-AMPK (#2535, 1:1000, Cell Signaling Technology, USA), AKT (#4685, 1:1000, Cell Signaling Technology, USA), p-AKT (#4060, 1:2000, Cell Signaling Technology, USA).
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!