Approximately 50 mg of lower right lung tissue samples were collected from the different groups of rats, followed by washing, homogenization, and lysing. After conventional protein extraction from lung tissue, total protein concentrations were measured. A total of 30 μg of total protein was then added to a one third volume of 4× sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer (cat. no. 30166428; Sinopharm Chemical Reagent Co., Ltd.), the protein sample was boiled for 10 minutes, and the protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transferring the separated proteins onto a polyvinylidene fluoride membrane (cat. no. IPVH00010; Emd Millipore BioTechniques Co., Ltd., Billerica, MA, USA), the protein blots were prepared according to conventional procedures of western blot analysis. After developing the protein blots in electrochemiluminescence reagent (cat. no. K-12043-D10; Advansta Inc., Menlo Park, CA, USA), the absorbance values of protein bands were scanned by a gel imaging system (LI-COR, Lincoln, NE, USA) to calculate the relative protein expression levels of MMPs and TIMPs according to the ratios of MMP and TIMP absorbance to β-actin absorbance.
Gel imaging system
Manufactured by LI COR
Sourced in United States
The Gel Imaging System is a laboratory equipment designed for capturing and analyzing gel-based images. It provides a platform for visualizing and documenting various types of gels, such as those used in electrophoresis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence detection kit, by Bio-Rad (1 mentions)
P erk, by R&D Systems (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Odyssey infrared scanner, by LI COR (1 mentions)
Goat anti rabbit igg, by Beyotime (1 mentions)
Hrp conjugated goat anti mouse igg, by Beyotime (1 mentions)
Ab81321, by Abcam (1 mentions)
Ab24121, by Abcam (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Af0003, by Beyotime (1 mentions)
3 protocols using gel imaging system
1
Lung Tissue MMP-TIMP Quantification
2
Western Blot Analysis of Apoptosis Signaling
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Total protein from the NRK-52E cells was extracted with radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific). Then, the proteins were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk for 2 h at room temperature. Next, the membranes were incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies. The protein expression levels were measured by an enhanced chemiluminescence detection kit (Bio‐Rad, Hercules, CA, USA) and the bands were observed using a gel imaging system (Odyssey, LI‐COR Biosciences). The primary antibodies used were as follows: anti-cleaved caspase3 (#9662; dilution: 1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-Bcl-2-associated X protein (Bax, #50599-2; dilution: 1:1000; Proteintech), p-JNK (#4668; dilution: 1:1000; Cell Signaling Technology), JNK (#9258; dilution: 1:1000; Cell Signaling Technology), p-P38 (#4511; dilution: 1:1000; Cell Signaling Technology), P38 (#8690; dilution: 1:1000; Cell Signaling Technology), p-ERK (#AF-1018; dilution:1:2000; R&D, Minnesota, MN, USA), ERK (#AF-1576-SP; dilution:1:1000; R&D), and anti-GAPDH (#60004-1; dilution: 1:8000; Proteintech).
3
Western Blot Analysis of Protein Signaling
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Cells were lysed in 2× Laemmli loading buffer and denatured at 95 °C for 5 min. Then proteins were separated on 10% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked in 5% BSA or non-fat dried milk (NFDM) in TBS with 0.1% Tween20 (TBST) at RT for 1 h, followed by incubation at 4 °C overnight with primary antibodies: STAT3 (CST, 9139 s, 1:1000), phospho-STAT3 (CST, 9145 s, 1:1000), ERK (CST, #4695, 1:2000), phospho-ERK-Thr202/Tyr204 (CST, #9101, 1:2000), AKT (CST, 4685 s, 1:2000), phospho-AKT-S473 (CST, 4060 s, 1:2000), NRP1 (Abcam, ab81321, 1:1000), PLAU (Abcam, ab24121, 1:1000), LRP1 (Abcam, ab92544, 1:1000), and β-actin (Beyotime, AF0003, 1:5000). After washed with TBST for three times, membranes were incubated with HRP-conjugated goat anti-mouse IgG (Beyotime, A0216, 1:1000) or goat anti-rabbit IgG (Beyotime, A0208, 1:1000) at RT for 1 h. After washing three times with TBST, blots were detected with Clarity Western ECL Substrate (Bio-Rad) through a Gel Imaging system (Tanon 6100 C) or an Odyssey infrared scanner (LICOR Bioscience).
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