Total proteins were isolated from the NP cells using RIPA buffer (Cell Signaling Technology, Inc.). The BCA Protein Assay kit (Pierce) was applied to measure the protein concentration, which was adjusted to a concentration of 6 µg/µl using 1X loading buffer and DEPC water. Electrophoresis was used to separate the samples using a 10% SDS-PAGE gel followed by transfer onto a polyvinylidene fluoride membrane (PVDF, Millipore). After blocking in 5% non-fat milk in PBST [0.1% Tween-20 in phosphate-buffered saline (PBS)] for 1 h, the membrane was incubated with the primary antibody overnight at 4°C, and subsequently incubated with secondary antibody [horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG, 1:2,000; sc-516102/sc-2357; Santa Cruz Biotechnology, Inc.] at room temperature for 2 h. Developer (EZ-ECL kit; Biological Industries BI) was used for development, and the gray value of the strips were analyzed and quantified using imageJ software (version 5.0; Bio-Rad). The antibodies utilized included anti-GAPDH (mouse; 1:1,000; LS-B1625; LifeSpan BioSciences, Inc.), anti-TLR4 (rabbit; 1:500; ab13556; Abcam), anti-IκBα (mouse; 1:1,000; #4814), anti-p-IκBα (mouse; 1:1,000; #9246), anti-p65 (rabbit; 1:1,000; #8242), anti-p-p65 (rabbit; 1:1,000; #3039) and anti-TIMP3 (rabbit; 1:1,000; #5673) (all from Cell Signaling Technology, Inc.).
Anti timp3
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-TIMP3 is a laboratory reagent produced by Cell Signaling Technology. It is an antibody that binds to and detects the TIMP3 protein. TIMP3 is an extracellular matrix metalloproteinase inhibitor that plays a role in regulating tissue remodeling and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Anti mmp2, by Cell Signaling Technology (2 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Anti p iκbα, by Cell Signaling Technology (1 mentions)
Anti p p65, by Cell Signaling Technology (1 mentions)
Sc 516102 sc 2357, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Anti tlr4, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Anti iκbα, by Abcam (1 mentions)
Anti atf3, by Abcam (1 mentions)
Sds polyacrylamide gel, by Bio-Rad (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Wet blotting, by Bio-Rad (1 mentions)
Enhanced chemiluminescence, by Bio-Rad (1 mentions)
Anti mmp9, by Cell Signaling Technology (1 mentions)
Anti annexin 5, by Cell Signaling Technology (1 mentions)
Anti gm130, by Cell Signaling Technology (1 mentions)
Anti mt1 mmp, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti timp3
1
Western Blot Analysis of Immune Signaling
2
Quantifying Glioma Cell Secretome Changes
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U87 and primary IC glioma cells were cultured in DMEM containing 0.4% FBS. Supernatant of JS-K treated cells was collected and frozen at −20 °C. Cells for whole cell lysates were cultured in 10% FBS. Equal amounts of protein (4 μg of supernatant, 20 μg of lysate) were applied on 10% SDS-polyacrylamide gels and electrophoresed (BioRad, Munich, Germany). Proteins were blotted on PVDF-membranes by wet blotting (BioRad, Munich, Germany). Epitopes were blocked with 5% non-fat milk in tris-buffered saline with 0.05% Tween20 for 1 h at room temperature (RT). Blots were incubated with primary antibodies anti-ATF3 (ab87213 1 : 1000 Abcam, Cambridge, UK), anti-MMP2 (#4022 1 : 1000 Cell Signaling Technology, Inc., Danvers, MA, USA), anti-MMP7 (#MAB9071 1 : 1000 R&D System, Inc., Minneapolis, USA), anti-MMP9 (#3852 1 : 1000 Cell Signaling Technology, Inc.) anti-TIMP3 (#D74B10 1 : 1000 Cell Signaling Technology, Inc.), anti-GAPDH (1 : 10000 Abcam) overnight at 4 °C. After incubation with secondary antibodies goat anti-rabbit/mouse (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at RT proteins were visualized by enhanced chemiluminescence (BioRad). For loading control, a Coomassie-stained SDS-polyacrylamide gel was used for whole protein in the supernatant and GAPDH was used for the whole cell lysate.
3
Western Blot Analysis of Protein Expression
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Protein lysates were prepared using RIPA lysis buffer (Thermo Fisher Scientific), and protein concentration was determined by BCA Protein Assay Kit (Thermo Fisher Scientific). Protein lysates were separated by SDS-PAGE and transferred onto PVDF membrane (Bio-Rad). After blocking, the blot was incubated with primary antibody at 4 °C overnight, followed by incubation with secondary antibody. Chemiluminescence signal was visualized using SuperSignal Pico PLUS chemiluminescent Substrate (Thermo Fisher Scientific). The following primary antibodies were used in this study: anti-GM130 (1:1000, #12480); anti-CD54 (1:1000, #4915); anti-Annexin V (1:1000, #8555); anti-CD9 (1:1000, #13174); anti-TIMP2 (1:1000, #5738); anti-TIMP3 (1:1000, #5673); anti-GAPDH (1:1000, #5174); anti-VEGFR2 (1:1000, #9698); anti-ERK (1:1000, #4695); anti-p-ERK (1:1000, #4370); anti-MMP2 (1:1000, #87809); anti-MT1-MMP (1:1000, #13130) antibodies were purchased from Cell Signaling technologies (Beverly, MA, USA).
4
Circadian Rhythm Protein Analysis
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The cells were lysed with RIPA lysis buffer (Millipore-Sigma, Billerica, MA, USA) containing protease inhibitors (Millipore-Sigma). Protein concentration was determined by BCA assays, and 30 µg cell lysates were resolved by SDS-PAGE on 4–12% gradient Bio-Tris gels, transferred to nitrocellulose membranes (Thermo Fisher Scientific), and probed with each of the following antibodies: anti-CLOCK, anti-BMAL1, anti-TIMP3, anti-C/EBP-α, anti-C/EBP-β, anti-TNF-α, anti-NF-κB, anti-phospho-NF-κB, and anti-I-κB (Cell Signaling Technology, Beverly, MA, USA); anti-MMP-1 (courteously provided by Prof. Jin Ho Chung, Seoul National University College of Medicine, Seoul, South Korea); and GAPDH and horseradish peroxidase–conjugated secondary anti-rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA). Western blotting luminol reagent (Santa Cruz Biotechnology) was used to develop the signals. For zymogram analysis, the cultured medium was harvested every 4 or 12 h after synchronization or UV irradiation and loaded on 10% Zymogram (gelatin) Protein Gels (Thermo Fisher Scientific). The gels were incubated at 37°C overnight and stained with 0.5% Coomassie blue (Millipore-Sigma) according to the manufacturer’s instructions.
5
Immunoblotting Targets in Cell Signaling
6
Western Blot Analysis of Immune and Metabolic Markers
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Protein preparation and Western blots were performed as described previously [22 (link)]. The following primary antibodies were used: anti-F4/80 (1:1000; Abcam), anti-α-SMA (1:1000; Sigma), anti-TIMP3 (1:1000; Cell Signaling Technology), anti-α-tubulin (1:1000; Cell Signaling Technology), anti-PGC-1β (1:1000; abcam), anti-G6PC (1:1000; Invitrogen).
7
Western Blot Analysis of TIMP-3 Protein
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For western blot analyses, cells were lysed with RIPA lysis buffer (50 mM Tris‐HCl, 1% NP‐40, 0.25% Na‐deoxycholate, 50 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mg/mL aprotinin, 1 mM Na3VO4, 1 mM NaF). Next, 25 mg of total protein was electrophoresed on a 12% SDS‐PAGE gel, transferred to a PVDF membrane (Amersham Pharmacia Life Science, USA), and blocked with 10% non‐fat milk in TBST. The PVDF membrane was incubated with anti‐TIMP‐3 (1:1000; Cell Signaling Technology, Inc., Beverly, MA, USA) and anti‐GAPDH antibody (Zhongshan Biological, Beijing, China) overnight at 4°C to confirm equivalent protein loading in each lane, followed by incubation with HRP‐conjugated goat‐anti‐rabbit IgG for one hour at room temperature, washed three times with TBST for 15 minutes each, and finally developed with ECL (Amersham Life Science) on X‐ray film.
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