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.
Mmp 2
Manufactured by Affinity Biosciences
Sourced in China
MMP-2 is a lab equipment product developed by Affinity Biosciences. It is used to measure the activity of the Matrix Metalloproteinase-2 enzyme. MMP-2 plays a role in the breakdown of extracellular matrix proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mmp 9, by Affinity Biosciences (5 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence detection kit, by Solarbio (1 mentions)
Tris buffered saline solution, by Solarbio (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Rottlerin, by Merck Group (1 mentions)
Eht1864, by Merck Group (1 mentions)
Snail, by Affinity Biosciences (1 mentions)
Sb239063, by Selleck Chemicals (1 mentions)
Tgf β, by Affinity Biosciences (1 mentions)
Collagen 1, by Affinity Biosciences (1 mentions)
Nf κb, by Affinity Biosciences (1 mentions)
Enhanced chemiluminescence ecl kit, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
P stat3, by Abmart (1 mentions)
Radioimmunoprecipitation assay (ripa), by Ipsen (1 mentions)
Hoxb5, by Abcam (1 mentions)
Bcl2 associated x (bax), by Affinity Biosciences (1 mentions)
β actin, by Abmart (1 mentions)
Bcl 2, by Affinity Biosciences (1 mentions)
6 protocols using mmp 2
1
Quantifying MMP-2 and MMP-9 Levels
2
Inhibition of Cell Migration Signaling
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BZN, EIPA (NHE1 inhibitor), EHT1864 (Rac1 inhibitor), rottlerin (PKC inhibitor), and pamoic acid disodium (PAM, ERK1/2 activator) were purchased from MCE, and SB239063 (p38 inhibitor) from Selleck. BZN (10 mM), EIPA (40 mM), EHT1864 (20 mM), rottlerin (20 mM), and PAM (30 mM) were all dissolved in DMSO (Sigma, CA, USA) and stored at −80 °C. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. All primary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA), except for antibodies against MMP-2, MMP-9, Snail, p-JNK1/2/3 (Thr183 + Tyr185) (Affinity Biosciences, Cincinnati, OH, USA), and β-actin (Zoonbio Biotechnology, Nanjing, China).
3
Immunohistochemical Analysis of Wound Tissue
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The available wound tissue sections were deparaffinized and rehydrated. Subsequently, 3% H2O2 in methanol was used for 10 min to block endogenous peroxidase activity. After boiling in 0.01 mol/L citric acid solution for 20 min to retrieve the antigen, normal goat serum was applied at 37 °C for 10 min. The sections were treated with the following primary antibodies: collagen I (Affinity, Changzhou, China), NF-κB (Affinity, Changzhou, China), MMP-2 (Affinity, Changzhou, China), MMP-9 (Affinity, Changzhou, China), and TGF-β (Affinity, Changzhou, China). After incubation for 1 h, sections were washed with phosphate-buffered saline (PBS) and incubated with biotinylated goat anti-mouse IgG antibody (Zhongshan Biology Technology Co., Ltd., Beijing, China) at 37 °C for 30 min. Following staining with 3, 3'-diaminobenzidine (DAB)/H2O2 and HE, sections were cleared and mounted for observation and analysis.
4
Quantifying Protein Expression Levels
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Cell protein samples were extracted, assessed (BCA kit, Beyotime, China), balanced, and denatured (with loading buffer, 100°C). Then the samples were added to a glue plate for electrophoresis and transferred to the membrane. After being sealed with milk, it was incubated with primary antibodies (MMP2, 1:1,000, Affinity, China; MMP9, 1:1,000, Affinity, China; CBX7, 1:1,000, Abcam, United Kingdom; CBX8, 1:1,000, Abcam, United Kingdom) at 4°C overnight and secondary antibody the next day. The band was exposed via the Enhanced Chemiluminescence (ECL) Kit (Beyotime) and quantified via the Image J Software (NIH, Bethesda, MD, United States) by a technician (blinded to the experimental groupings) for statistical analysis.
5
Investigating HOXB5 and JAK/STAT Signaling in Osteosarcoma
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Osteosarcoma cells were lysed using RIPA (Epizyme, China) with PMSF (Epizyme, China), and the concentration of the protein lysates was measured using the BCA test. Proteins with different molecular masses were separated using SDS‒PAGE, transferred to PVDF membranes, and after 1 h of blocking, they were incubated with the indicated primary antibodies overnight at 4 °C. The next day, the membrane was treated for 1 h with the relevant secondary antibodies, after which the stained bands were visualised with an ECL system and quantified with ImageJ. Primary antibodies against the following proteins were used: HOXB5 (1:1000, Abcam); Bcl-2, Bax, MMP2, MMP9 (1:1000, Affinity); JAK2, p-JAK2, STAT3, p-STAT3, β-actin (1:5000, Abmart).
6
Protein Expression Analysis in Tumor Cells
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Protein was extracted from tumour tissues of nude mice and G401 cells treated with DCH and lysed using RIPA buffer containing 1% protease inhibitor, and the protein concentration was determined by BCA assay. An exact amount of protein (20 μg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Microwell, USA). The membranes were then blocked with 5% skim milk for 1 h and incubated with primary antibody overnight at 4°C. The primary antibodies were as follows: MMP2 (1 : 1000, Affinity Biosciences, Cat No. #AF5330), MMP9 (1 : 1000, Affinity Biosciences, Cat No. #AF5228), E-cadherin (1 : 1000, ZENBIO, Cat No. 201283), ZO-1 (1 : 1000, Affinity Biosciences, Cat No. #AF5145), α-SMA (1 : 1000, ZENBIO, Cat No. 380653), N-cadherin (1 : 1000, Proteintech, Cat No. 66219-1-Ig), vimentin (1 : 1000, ZENBIO, Cat No. R22775), and GAPDH (1 : 5000, ZENBIO, Cat No. 200306-7E4). The next day, the samples were washed 3 times using TBST, incubated with secondary antibodies at room temperature for 1 h, washed 3 times using TBST, and imaged using chemiluminescence, employing statistical grey values from Image Lab.
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