Levels of the MMP-2 and MMP-9 proteins were examined using Western blotting. Briefly, proteins were extracted from HK-2 cells and PMNs, separated on 10% SDS-PAGE gels, and transferred onto nitrocellulose membranes (Millipore, Burlington, MA). The membranes were blocked with 5% nonfat milk in a Tris-buffered saline solution (Solarbio Life Science, Beijing, China) and incubated with the following rabbit primary antibodies: MMP-9 (1:1,000; Affinity Biosciences, Zhenjiang, China) and MMP-2 (1:1,000; Affinity Biosciences, Zhenjiang, China). The membranes were then incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (1:3,000; ZSGB-BIO, Wuhan, China). The specific bands were developed and visualized using an enhanced chemiluminescence detection kit (Solarbio Life Science, Beijing, China). Images were analyzed using ImageJ software.
Enhanced chemiluminescence detection kit
Manufactured by Solarbio
Sourced in China
The Enhanced Chemiluminescence Detection Kit is a laboratory equipment designed to detect and quantify proteins in Western blot analysis. It utilizes a chemiluminescent substrate to generate a luminescent signal proportional to the amount of target protein present in the sample. The kit provides a sensitive and reliable method for protein detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (1 mentions)
Mmp 9, by Affinity Biosciences (1 mentions)
Tris buffered saline solution, by Solarbio (1 mentions)
Mmp 2, by Affinity Biosciences (1 mentions)
Ripa reagent, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Beyotime (1 mentions)
Ab6721, by Abcam (1 mentions)
Bicinchoninic acid assay kit, by Solarbio (1 mentions)
Ab214424, by Abcam (1 mentions)
Ab22555, by Abcam (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Ab179483, by Abcam (1 mentions)
Ab92498, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Ab220166, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
5 protocols using enhanced chemiluminescence detection kit
1
Quantifying MMP-2 and MMP-9 Levels
2
Western Blot Analysis of STAT Proteins
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The western blot (WB) assay was used to detect the expression changes of STAT1 and STAT3 in rat lung tissues. Stored rat lung tissues were lysed using RIPA reagent (Beyotime, China), prepared and detected using protein gel electrophoresis. Proteins were transferred to a PVDF membrane, blocked, and incubated with diluted primary antibody, and then secondary antibody. The primary and secondary antibodies were incubated for 1 h and 1–2 h, respectively. Protein visualization was conducted using an enhanced chemiluminescence detection kit (Solarbio, China). The Image J 1.8.0 software was used to determine the optical density values of protein bands and the internal control β-actin, and the relative density of the protein in each group was calculated from the ratio of the two. Immunoblotting was performed and blots were probed with antibodies against IL-6, JAK1, STAT3, SOCS3, and β-actin (CST, USA).
3
Protein Expression Analysis in HESC and Endometriosis
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Protein extraction from HESCs or endometriotic lesions was carried out using RIPA lysis buffer (Solarbio), and a bicinchoninic acid assay kit (Solarbio) was utilized for measuring the protein concentration. Protein samples (20 μg) were dissolved with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto polyvinylidene fluoride membranes (Beyotime), and blocked with 5% defatted milk. Next, the membranes were incubated overnight with anti-HIF1AN (ab92498, 1:5,000), anti-HIF1A (ab179483, 1:1,000), anti-VEGF (ab214424, 1:1,000), and anti-GAPDH (ab22555, 1:1,000) primary antibodies (all from Abcam) at 4℃, and then incubated at room temperature with the secondary antibody (ab6721, 1:2,000, Abcam) for 2 h. Protein bands were visualized with the enhanced chemiluminescence detection kit (Solarbio) and quantified with ImageJ software (NIH).
4
Skeletal Muscle Protein Expression Analysis
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After 12 weeks of different light treatments, the mice in each group (n = 3) were euthanized by cervical dislocation, and skeletal muscle (gastrocnemius muscle) tissues were isolated. The total protein of skeletal muscle tissues were extracted by using RIPA buffer (Beyotime Institute of Biotechnology). Protein quantification was performed using the BCA kit according to the instruction. The protein (15–30 μg) were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. After blocking with 5% nonfat milk for 2 h, the membranes were incubated with target primary antibodies (including anti-IRS-1, anti-p-IRS-1, anti-Akt, and anti-p-Akt) at 4 °C overnight. Then the membranes were incubated with corresponding secondary antibody at room temperature for 1 h. The protein bands were exposed by Bio-Rad exposure instrument using an enhanced chemiluminescence detection kit (Solarbio).
5
Western Blot Protein Analysis Protocol
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Briefly, after cell lysis with RIPA + a protease inhibitor, centrifugation of the
lysate was performed at 4°C for 10 min at 12 000 r/min. The obtained protein
samples were quantitated with a BCA kit (Thermo Fisher Scientific, MA, USA),
boiled at 95°C, and sequentially separated by SDS-PAGE, which were loaded to
nitrocellulose membranes. Then, membranes were silked in 5% skimmed milk.
Membranes were reacted with primary antibodies (ID4 [ab220166, 1:1000, Abcam,
Cambridge, UK] and GAPDH [ab181602, 1:10 000, Abcam, Cambridge, UK]) overnight.
Later, they were cultured with horseradish peroxidase (HRP)-conjugated secondary
antibody goat anti-rabbit IgG (ab6721, 1:3000, Abcam, Cambridge, UK) for
120 min. At last, an enhanced chemiluminescence detection Kit (Solarbio,
Beijing, China) was used for protein band visualization.
lysate was performed at 4°C for 10 min at 12 000 r/min. The obtained protein
samples were quantitated with a BCA kit (Thermo Fisher Scientific, MA, USA),
boiled at 95°C, and sequentially separated by SDS-PAGE, which were loaded to
nitrocellulose membranes. Then, membranes were silked in 5% skimmed milk.
Membranes were reacted with primary antibodies (ID4 [ab220166, 1:1000, Abcam,
Cambridge, UK] and GAPDH [ab181602, 1:10 000, Abcam, Cambridge, UK]) overnight.
Later, they were cultured with horseradish peroxidase (HRP)-conjugated secondary
antibody goat anti-rabbit IgG (ab6721, 1:3000, Abcam, Cambridge, UK) for
120 min. At last, an enhanced chemiluminescence detection Kit (Solarbio,
Beijing, China) was used for protein band visualization.
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