Total cellular protein was extracted with RIPA buffer (Beyotime). Cell lysates (20 μg per sample) were subjected to 10% SDS-PAGE, transferred to a PVDF membrane (Merck Millipore, Billerica, USA), blocked with 5% fat-free milk, and incubated with primary antibody (1:1000) at 4°C overnight. After that, the membrane was incubated with HRP-conjugated secondary antibodies (Beyotime). The bands were detected with a Bio‐Rad Imaging System (Bio-Rad, Hercules, USA) after incubation with ECL substrate. The following primary antibodies were used: CTSK (Beyotime), MMP-9 (Beyotime), nuclear factor of activated T cells 1 (NFATc1; Abcam, Cambridge, UK), GAPDH (Cell Signaling Technology, Boston, USA), p38 (Cell Signaling Technology), p-p38 (Cell Signaling Technology), ERK (Cell Signaling Technology), p-ERK (Cell Signaling Technology), JNK (Cell Signaling Technology), p-JNK (Cell Signaling Technology), TAK1 (Beyotime), p-TAK1 (Beyotime), IKK (Beyotime), p-IKK (Beyotime), IκBα (Beyotime), p-IκBα (Beyotime), p65 (Beyotime), and p-p65 (Beyotime). GAPDH was used as an internal control.
Mmp 9
Manufactured by Beyotime
Sourced in China
MMP-9 is a laboratory equipment used for the detection and quantification of matrix metalloproteinase-9 (MMP-9) in various biological samples. MMP-9 is an enzyme involved in the breakdown of extracellular matrix proteins, which play a crucial role in various physiological and pathological processes. The core function of this equipment is to provide accurate and reliable measurements of MMP-9 levels, enabling researchers and scientists to study its role in different research applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Beyotime (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
P ikk, by Beyotime (1 mentions)
P iκbα, by Beyotime (1 mentions)
Nfatc1, by Abcam (1 mentions)
E cadherin, by Beyotime (1 mentions)
Vimentin, by Beyotime (1 mentions)
β actin, by Beyotime (1 mentions)
Pnf κb, by Beyotime (1 mentions)
Nf κb, by Beyotime (1 mentions)
Hrp labeled goat anti rabbit igg h l, by Beyotime (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
High glucose dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Proliferating cell nuclear antigen (pcna), by Beyotime (1 mentions)
4 protocols using mmp 9
1
Protein Extraction and Western Blot Analysis
2
Immunofluorescence detection of MMP-1 and MMP-9
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IF staining was used to detect the specific expression of MMP-1 and MMP-9 in the cells. We treated HaCaT cells with 4% paraformaldehyde fixation, 0.2% Triton X-100 permeabilization, and 1% BSA closure and finally added the following primary antibodies: MMP-1 (1:200; Cat. No. AF0231; Beyotime) and MMP-9 (1:200; Cat. No. K001664P; Solarbio) and then incubated the cells overnight at 4°C. The following day, goat anti-rabbit IgG-FITC secondary antibody (1:500; Cat. No. A0562; Beyotime) was added, and finally the nuclei were stained with DAPI, and images were obtained.
3
Western Blot Analysis of Cellular Proteins
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RIPA buffer (Beyotime, China) was used to lyse cells to obtain total protein, and the protein concentration was determined by BCA method. The same amount of samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein was transferred to the PVDF membrane (Millipore, Burlington, United States). After blocking with 5% skim milk powder (Beyotime, China) for 2 h at room temperature, the membrane and antibody were incubated overnight at 4°C. The primary antibodies were as follows: AMPK, NF-κB, p-NF-κB, MMP9, MMP2, TIMP-2, E-cadherin, Vimentin and β-actin (Beyotime, China). Then incubated with HRP-labeled Goat Anti-Rabbit IgG (H + L) (Beyotime, China) at room temperature for 2 h. Protein bands were detected by enhanced chemiluminescence (Tanon chemiluminescent gel imaging system, Tanon-5200Multi, China).
4
Alkaloid Extraction and Identification from Plumula Nelumbinis
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The separation, extraction, and identification of the total alkaloids from Plumula Nelumbinis (TAPN), neferine, liensinine, and isoliensinine were performed as described in our previous study (Liu et al., 2009 (link); Jiang et al., 2018a (link); Chen et al., 2020 (link)). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (C6164, Darmstadt, Germany). High-glucose Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific). Primary antibodies against collagen1, collagen3, MMP2, MMP9, PCNA, PIM1, p-SRC, and SRC were purchased from Beyotime (Shanghai, China). α-Tubulin was purchased from Santa Cruz Biotechnology (CA, United States).
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