Chondrocytes were lysed by radio immunoprecipitation assay (RIPA) lysis buffer with phenylmethylsulfonyl fluoride (PMSF) and a phosphatase inhibitor. The concentration of proteins was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Beyotime Biotechnology, China). The proteins (30 µg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to polyvinylidenedifluoride (PVDF) membranes. The membranes were blocked in a nonfat dry milk solution for 1 hour. Then, the membranes were incubated overnight at 4°C with anti-AMPK (CST, #2532, 1:1000 dilution), anti-p-AMPK (CST, #2535, 1:1000 dilution), anti-mTOR (CST, #2983, 1:1000 dilution), anti-p-mTOR (CST, #5536, 1:1000 dilution), anti-Beclin-1 (Abcam, ab207612, 1:1000 dilution), anti-ULK1 (Abcam, ab133747, 1:1000 dilution), anti-LC3 II (Abcam, ab48394, 1:1000 dilution), anti-P62 (Abcam, ab56416, 1:2000 dilution), anti-Bcl2 (Abcam, ab194583, 1:2000 dilution) and anti-Bax (Abcam, ab32503, 1:1000 dilution) primary antibodies in dilution buffer. Next, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (Zhongshan Golden Bridge Biotechnology, 1:5000 dilution) at room temperature for 1 hour. Then, the membranes were developed using the enhanced chemiluminescence substrate LumiGLO (Millipore, Bedford, MA, USA).
Lumiglo
Manufactured by Merck Group
Sourced in United States
LumiGLO is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It produces a luminescent signal when interacting with the target protein, which can be captured on photographic film or a digital imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ab48394, by Abcam (2 mentions)
Ab56416, by Abcam (2 mentions)
Celllytic nuclear extraction kit, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Ab207612, by Abcam (1 mentions)
Ab194583, by Abcam (1 mentions)
Ab133747, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Bovine serum albumin reagent, by Beyotime (1 mentions)
Gel pro analyzer 4, by Bio-Rad (1 mentions)
Anti ulk1, by Cell Signaling Technology (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Anti β actin, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Hrp conjugated anti rabbit igg, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
5 protocols using lumiglo
1
Western Blot Analysis of Chondrocyte Proteins
2
Nuclear Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were placed on ice immediately following treatment and washed with ice-cold Hank’s Balanced Salt Solution. Nuclear proteins were prepared using the CellLytic NuCLEAR extraction kit (Sigma-Aldrich). All the wash buffers and final resuspension buffer included a 1× protease inhibitor cocktail (Pierce, Rockford, IL, USA), NaF (5 mM), and Na3VO4 (200 mM). The protein concentration of the lysate was measured using the BCA protein assay kit (Thermo Fisher). Nuclear or total cell proteins were resolved on 8 to 12% SDS-PAGE and transferred by electroblotting to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% bovine serum albumin reagent (Beyotime, Jiangsu, China) and incubated overnight at 4 °C with primary antibody dilution buffer (the dilution followed the specification; Abcam) and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000) for 2 h. Afterward, the membranes were developed using the enhanced chemiluminescence substrate LumiGLO (Millipore, Bedford, MA, USA) and exposed to X-ray film. The bands were analyzed with Gel-Pro Analyzer 4.0 (Bio-Rad, Hercules, CA, USA).
3
Nuclear 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 were placed on ice immediately following treatment and washed with ice‐cold Hank's balanced salt solution. Nuclear proteins were prepared using the CellLytic NuCLEAR extraction kit (Sigma‐Aldrich, St. Louis, MO). All the wash buffers and final resuspension buffer included 1× protease inhibitor cocktail (Pierce, Rockford, IL), NaF (5 mM), and Na3VO4 (200 mM). The protein concentration of the lysate was measured using the BCA protein assay kit (Thermo Fisher Scientific, Boston, MA). Nuclear or total cell proteins were resolved on 8% to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred by electroblotting to nitrocellulose membranes (Bio‐Rad, Hercules, CA). The membranes were blocked in nonfat dry milk solution and incubated overnight at 4°C with ColII (1:1000), aggrecan (1:1000), Runx2 (1:1000), ColX (1:1000), p65 (1:1000), p50 (1:1000), p52 (1:500), RelB (1:500), cRel (1:1000), and NICD1 (1:1000) primary antibody dilution buffer and then incubated with horseradish peroxidase‐conjugated anti‐rabbit IgG (1:5000) for 2 hours. Afterwards, the membranes were developed using the enhanced chemiluminescence substrate LumiGLO (Millipore, Bedford, MA) and exposed to X‐ray film. The bands were analyzed with Gel‐Pro Analyzer 4.0.
4
Western Blot Analysis of Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All proteins were extracted using a radio immunoprecipitation assay (RIPA) lysis buffer with protease and phosphatase inhibitors. The protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Protein samples were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk solution for 1 h at room temperature. The membranes were then probed with anti-PPARγ (sc-7273, 1:200, Santa), anti-mTOR (#2983, 1:1000; Cell Signaling Technology), p-mTOR (#5536, 1:1000; Cell Signaling Technology), anti-ULK1 (#8054, 1:1000; Cell Signaling Technology), anti-LC3II (ab48394, 1:2000, Abcam), anti-P62 (ab56416, 1:2000; Abcam) and anti-β-actin (1:2000; Zhongshan Golden Bridge Biotechnology) overnight at 4 °C. The following day, membranes were washed three times with TBST and incubated with goat anti-rabbit or goat anti-mouse antibody IgG (H + L)-HRP (1:5000; Wuhan Sanying) for 1 h at room temperature. Finally, the membranes were visualized using the enhanced chemiluminescence substrate LumiGLO (Millipore, MA, USA).
5
Western blot analysis of DNA damage response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The SCNs were lysed with a cell lysate containing PMSF and phosphatase inhibitors. The cell lysates were collected and centrifuged at 14,000 rpm for 5 min. The supernatants were taken and the protein concentration was determined using a BCA kit (Beyotime Biotechnology, China). The protein sample was boiled for 10 min before loading, and then electrophoresed using 10% SDS gel, transferred to a PVDF membrane, blocked with 5% skim milk at room temperature for 1 h, and then the membranes were incubated overnight at 4°C with anti-PARP1(Abcam, ab151794, 1:1,000 dilution), anti-PAR(CST, #83732, 1:1,000 dilution), anti-γH2AX(CST, #2577, 1:1,000 dilution), anti-GAPDH(CST, #5174, 1:1,000 dilution), anti-p-AMPK (CST, #2535, 1:1,000 dilution), and anti-AMPK (CST, #2532, 1:1000 dilution) primary antibodies in dilution buffer. The cells were incubated with the horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Beyotime Biotechnology, A0216, 1:1,000 dilution). The membranes were developed using the enhanced chemiluminescence substrate LumiGLO (Millipore, Bedford, MA, 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!