Treated cells were lysed with RIPA lysis buffer (Bioswamp), and the protein concentrations in the lysates were measured with a BCA protein assay kit (Bioswamp). Equal amounts of proteins in each sample were loaded onto gels and subjected to SDS-PAGE. The separated proteins on the gels were then transferred to nitrocellulose membranes. Each immunoblot was blocked with 5% nonfat milk in TBST for 2 h prior to an overnight incubation at 4 °C with specific antibodies. The following primary antibodies were used for Western blotting: Bcl-2 (Proteintech, 60178-1-Ig), Bax (Proteintech, 50599-2-Ig), cleaved caspase-3 (Cell Signaling Technology, 9664), LC3B (Bioswamp, MAB43969), Beclin 1 (Proteintech, 66665-1-Ig), Parkin (Proteintech, 14060-1-AP), P62 (Proteintech, 18420-1-AP), Tim23 (Proteintech, 11123-1-AP), CoxIV (Cell Signaling Technology, 4844) and NDP52 (Cell Signaling Technology, 60732). The blots were washed with TBST three times for 15 min each and then incubated with secondary antibodies for 2 h at RT. Finally, enhanced chemiluminescence reagent was used to visualize the bands, and the intensities were determined to further evaluate the levels of each protein.
Tim23
Manufactured by Proteintech
Sourced in China
The TIM23 is a laboratory equipment product that serves as a key component in protein transport and import processes. It functions as a translocase, facilitating the translocation of proteins across the mitochondrial inner membrane. The TIM23 complex plays a crucial role in the import and sorting of nuclear-encoded proteins destined for the mitochondria.
Automatically generated - may contain errors
Lab products found in correlation
Tom20, by Proteintech (3 mentions)
Cox 4, by Cell Signaling Technology (2 mentions)
Parkin, by Proteintech (2 mentions)
Ndp52, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Lc3a b, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Bioswamp (1 mentions)
Bca protein assay kit, by Bioswamp (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
Beclin 1, by Proteintech (1 mentions)
Bcl 2, by Proteintech (1 mentions)
α tubulin, by Merck Group (1 mentions)
Ecl blotting detection reagents, by Thermo Fisher Scientific (1 mentions)
Actin, by Proteintech (1 mentions)
Parkin, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor mixture, by Roche (1 mentions)
Calnexin, by Proteintech (1 mentions)
Coralite594, by Proteintech (1 mentions)
Anti flag, by Proteintech (1 mentions)
8 protocols using tim23
1
Protein Expression Analysis of Apoptosis and Autophagy
2
Whole Cell Protein Extraction and Western Blot
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Cells were lysed in ice-cold whole cell extract buffer (50 mM TRIS-HCl, pH 8.0, 4 M urea and 1% Triton X-100), supplemented with complete protease inhibitor mixture (Roche Diagnostics, 04693132001). The whole cell extracts were resolved by SDS-PAGE gel electrophoresis, and transferred to nitrocellulose membranes. Membranes were probed with the indicated primary antibodies followed by appropriate HRP-conjugated secondary antibodies (KPL). Protein bands were visualized using ECL Blotting Detection Reagents (Thermo Scientific, 32106). Primary antibodies used for western blotting were as follows: Parkin (Cell Signaling, 4211), LC3B (Sigma, L7543), MFN1 (Abcam, ab57602), TIM23 (Proteintech, 11123-1-AP), COXIV (Cell Signaling, 4844), C-III core 1 (Invitrogen, 459140), p62 (MBL, PM045), cleaved Caspase-3 (Cell signaling Technology, 9664), cleaved PARP (Cell signaling Technology, 5625), Actin (Proteintech, 60008-1-IG), α-Tubulin (Sigma, T9026).
3
Cell Culture and Immunoblotting Protocol
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Human embryonic kidney (HEK293T), human liver cancer (HepG2), and African green monkey kidney (Vero) cells were grown and maintained in Dulbecco’s Modified Eagle Medium (DMEM, high glucose, HyClone) containing 10% heat-inactivated FBS (BBI), 100 U/ml of ampicillin, and 100 g/ml of streptomycin (Sangon). Anti-Flag, Cox IV, calnexin, Histone, GM130, TIM23, TOM20, P62, B23, GAPDH, and CoraLite 594 or 488-conjugated IgG secondary antibodies were obtained from Proteintech (Rosemont, IL, United States). HRP-labeled goat anti-mouse or rabbit IgG were purchased from Nachuan Bio, and anti-LC-3b antibody was from CST.
4
SARS-CoV-2 Protein Localization and Interaction
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The Nsp1–10 and Nsp12–16 fragments were obtained from the SARS-CoV-2 (MN908947.3) cDNA provided by Prof. Ke Peng (Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China) (Li et al., 2020 (link)). The Nsp1–10, Nsp12–16, and N- and C-terminal domains of Nsp8 fragments were cloned into pCMV-Flag vector. And the full-lenth, N- and C-terminal domains of Nsp8 were cloned into pCMV-GFP vector to generate pCMV-GFP-Nsp8, pCMV-GFP-Nsp8-N and pCMV-GFP-Nsp8-C plasmids. Flag-Nsp8 was cloned into pCDH-puro-EGFP to generate the pCDH-Nsp8-puro-EGFP plasmid. Supplementary Table S1 lists the primers used in plasmid construction. Mito-DsRed2-EGFP (Cat#P4954) and mCherry-EGFP-LC3 (Cat#P0446) plasmids were purchased from Miaoling Plasmid Sharing Platform (Wuhan, China). EGFP-LC3 was cloned by mutation of mCherry-EGFP-LC3. The following primary antibodies were used: TOM20 (Beyotime, AF1717); SQSTM1/p62 (Beyotime, AF5312); Beclin 1 (Beyotime, AF5123); ATG5 (Beyotime, AF2269); LC3A/B (Cell Signaling Technology, 12741S); anti-Mouse IgG 647 (Invitrogen, A31571); anti-Mouse IgG594 (Invitrogen, A21203); Flag (Proteintech, 20543-1-AP); β-Actin (Proteintech, 66009-1-Ig); Cytochrome c (Proteintech, 10993-1-AP); COXII (Proteintech, 55070-1-AP); TIM23 (Proteintech, 11123-1-AP); SARS-CoV-2-NP (Sino Biological, 40143-MM05).
5
Comprehensive Cardiac Protein Analysis
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Heart tissues or cultured cells were sonicated in RIPA lysis buffer and homogenized. The debris was removed and the supernatant was obtained after centrifugation at 12,000 g for 10 min at 4°C. About 30–50 μg proteins was loaded for electrophoresis, and probed with primary antibodies against collagen I (1:1000; No.14695-1-AP; Proteintech Co., Wuhan, China), α-SMA (1:1000; No.14395-1-AP; Proteintech Co.), TGF-β (1:1000; No.21898-1-AP; Proteintech Co.), LC3 I/II (1:1000; No.14600-1-AP; Proteintech Co., Wuhan, China), TIM23 (1:1000; No.11123-1-AP; Proteintech Co., Wuhan, China), TOM20 (1:1000; No.66777-1-Ig; Proteintech Co., Wuhan, China), OPTN (1:1000; No.10837-1-AP; Proteintech Co., Wuhan, China), COX4 (1:1000; No.11242-1-AP; Proteintech Co., Wuhan, China). GAPDH (1:1000, AF0006; Beyotime Biotechnology Co., Shanghai, China) was used as internal control. Images were analyzed using the Image-Pro Plus software.
6
Protein Expression Analysis of Rabbit Cartilage
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After the second behavioral assessment ended, the rabbits were sacrificed, and the cartilage was dissected with a blade. The protein concentrations of the cartilage samples were quantified with a BCA kit, the proteins were denatured, and protein expression levels were ultimately analyzed via Western blotting. Primary antibodies against Pink1 (Proteintech, Wuhan, China), Parkin (Proteintech, Wuhan, China), LC3 (MBL, Beijing, China), TOM20 (Proteintech, Wuhan, China), TIM23 (Proteintech, Wuhan, China), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, Cambridge, UK) as well as horseradish peroxidase-labeled secondary antibodies (Zhongshanjinqiao, Beijing, China) were used for Western blotting. The gray value of each band was analyzed by ImageJ.
7
Protein Expression Analysis in C2C12 Cells
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Western blot analysis was conducted to detect the expression of TIM23, LC3, p62, Bax, Bcl-2, P53, Nrf2, HO-1, Pink1, Parkin and β-actin protein in C2C12 myoblasts. LC3 rabbit monoclonal antibody and p62 rabbit polyclonal antibody (1:1000 dilution) were purchased from Cell Signaling (Beverly, MA, USA). Bax, Bcl-2, Nrf2, HO-1 and Parkin rabbit monoclonal antibodies (1:1000) were purchased from Abcam. Pink1 rabbit polyclonal antibody (1:1000) was purchased from ABclonal Technology. TIM23 and P53 rabbit polyclonal antibodies (1:1000) were purchased from Proteintech. β-actin mouse monoclonal antibody (1:2000) was purchased from Sungene. RIPA lysis buffer with 1% phosphatase inhibitor cocktail and 1 mM PMSF was used for the whole-cell lysates. Protein concentrations were detected via the BCA method (Biomed, Beijing, China) according to the manufacturer’s instructions. Equal amounts of protein were separated via 12% SDS-PAGE, transferred to PVDF membranes, and subsequently detected using various primary antibodies. The membranes were incubated for 1.5 h at room temperature with the appropriate secondary HRP-conjugated antibody (1:5000; Tianjin Sungene Biotech) and visualized using an ECL detection kit (Millipore Corporation, Billerica, MA, USA). The densities of specific bands were quantified using ImageJ software.
8
Immunoblot Analysis of Mitochondrial Proteins
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