The cells were lysed using EzRIPA buffer (ATTO, Tokyo, Japan). The lysate protein concentration was quantified by a Bradford assay (BioRad, Hercules, CA, USA) and measured using a BioTek Epoch Microplate Reader. Twenty micrograms of protein were subjected to 10%‐15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Amersham, GE Healthcare, Barcelona, Spain) using the electrophoretic method. The membrane was blocked by a PBS‐T solution which contained 5% skim milk for 60 minutes at room temperature. The primary antibodies, PINK1 (Novus Biologicals, Littleton, CO USA, #NB100‐493), phosphorylated AMPK (Cell Signaling, Boston, MA, USA, #2531), MDR1 (Santa Cruz biotechnology, CA, USA, #Sc‐55510) and β‐actin (Cell Signaling, Boston, MA, USA, #4967) were diluted 1:1000 in blocking solution (PBS‐T with 4% BSA) and incubated overnight at 4°C. The secondary HRP‐conjugated anti–rabbit (Santa Cruz Biotechnology) and anti–mouse (Santa Cruz Biotechnology) antibodies were used at a dilution of 1:1000 in blocking solution for 2 hours. Protein expression was detected by a chemiluminescence imaging system (ATTO) after spreading the Luminata Forte Western HRP Substrate (Millipore).
Pink1
Manufactured by Novus Biologicals
Sourced in United States
PINK1 is a recombinant protein that can be used for research purposes. It is a serine/threonine-protein kinase that plays a role in the regulation of mitochondrial function and dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Tom20, by Santa Cruz Biotechnology (3 mentions)
Cox 4, by Cell Signaling Technology (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Parkin, by Cell Signaling Technology (3 mentions)
Bradford assay, by Bio-Rad (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Parkin, by Santa Cruz Biotechnology (2 mentions)
Ab110413, by Abcam (2 mentions)
Lamp1, by Santa Cruz Biotechnology (2 mentions)
Dharmafect 1, by Thermo Fisher Scientific (2 mentions)
Evans blue, by Merck Group (2 mentions)
Lysotracker blue, by Thermo Fisher Scientific (2 mentions)
α actinin, by Cell Signaling Technology (2 mentions)
Rhogdi, by Cell Signaling Technology (2 mentions)
Lamin a c, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Lonp1, by Merck Group (2 mentions)
Phopsho p62, by Cell Signaling Technology (2 mentions)
C300 imaging system, by Azure Biosystems (2 mentions)
20 protocols using pink1
1
Western Blot Analysis of Protein Targets
2
Comprehensive Protein Analysis in Stem Cells
3
Comprehensive Mitophagy Regulation Assay
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The primary antibodies used were RhoA (#2117, for WB), Parkin (#2132), COX-IV (#11967), VDAC (#4661), lamin A/C (#2032), Rho-GDI (#2564), HA (#3724), PKD (#90039), P-PKD S916 (#2051), GAPDH (#2118), α-actinin (#3134) and LC3B (#3868) from Cell Signaling Technology; PINK1 from Novus Biologicals (#NB600-973); RhoA (SC-418, for IP) and ubiquitin (SC-8017) from Santa Cruz Biotechnology; miniSOG from Kerafast (#EFH004). Horseradish peroxidase (HRP)-conjugated secondary antibodies for WB, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), cycloheximide (CHX), MG-132, CID755673, Bafilomycin A1, Evans blue and triphenyltetrazolium chloride (TTC) were purchased from MilliporeSigma. Y-27632 was purchased from Cell Signaling Technology. DharmaFECT-1 and LysoTracker Blue were purchased from Thermo Fisher Scientific. C3 exoenzyme was purchased from Cytoskeletton, Inc.
4
Western Blot Analysis of Spinal Cord Proteins
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According to the method of our previous studies [28 (link),32 (link)], Western blot analysis was performed. In brief, the ipsilateral dorsal horns of the L5 spinal cord region were collected and used for the experiment. After homogenizing the tissue in PRO-PREPTM (iNtRON Biotechnology, 17081, Kirkland, Washington, USA) and PNPP (Sigma, N9389, St. Louis, MO, USA), lysates were centrifuged. Protein was quantified by Bradford assay (BioRad, # 5000002, Hercules, CA, USA) using only supernatant and 20 μg of protein were used. Proteins were transferred using NC membrane and all blots were blocked with 5% skim milk (BD Biosciences, 90002-594, Franklin Lakes, NJ, USA). p-p66shc (Enzo Life Sciences, ALX-B04-358-C100, 1:1000, Lausen, Switzerland), total shc (BD Biosciences, #6108780, 1:1000), cleaved Caspase-3 (Cell signaling, 9661T, 1:1000), p62 (Sigma, P0067, 1:1000) and PINK1 (Novus Biologicals, NBP2-36488, 1:1000, Centennial, CO, USA) were used as primary antibodies. Visual confirmation of the band was developed using ClarityTM Western ECL Substrate (BioRad, 170-5061).
5
Autophagy Flux Analysis Protocol
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To determine autophagy flux, proteins were harvested using radio immunoprecipitation (RIPA) buffer with protease and phosphatase inhibitors. Immunoblotting was performed using β-actin, VDAC, LC3B (Cell Signaling-Cat: 3700, 4866, 2775), PINK1 (Novus Biologicals-Cat: BC100494) MFN2, ATPase5 (AbCam-Cat: ab56889, ab14748) and Tom20 (Santa Cruz-Cat: sc17746). Images were collected using an Odyssey imager (LI-COR). Densitometry was analyzed using Image J software.
6
Protein Extraction and Western Blot Analysis
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Protein was extracted from lung homogenate or cell lysate using RIPA buffer or Mitochondria Isolation Kit (Pierce, Rockford, IL), separated on NuPAGE 4–12% Bis-Tris gels (Invitrogen) and transferred to PVDF membranes by electroblotting. Primary antibodies used were PINK1 (Novus, Littleton, CO), β-actin (Sigma), COX-4 (Santa Cruz Biotechnology, Dallas, TX), phospho-DRP1 (Ser 616; Cell Signaling, Beverly, MA), VDAC (Cell Signaling). Secondary antibodies were HRP-conjugated goat anti-rabbit or goat anti-mouse (Santa Cruz).
7
Protein Isolation and Western Blot Analysis
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PT cells were lysed for protein isolation using; 25 mM Tris-HCl pH 8, 1 mM EDTA, 150 mM NaCl, 1% NP-40, 1 mM Na3VO4, and protease inhibitor cocktails (Sigma). The tissue lysis buffer additionally contained 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. Proteins of both tissue and cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. All primary antibodies were dissolved in 5% BSA solution and incubated with membranes overnight at 4 °C. The following primary antibodies were used: Pink1 (Novus), LC3A (CST), Pgc1α (Merck), cleaved caspase 9 (CST), phospho-Smad3 (CST), total OXPHOS (ab110413, Abcam), Polγ (Santa Cruz), Tom20 (Santa Cruz), α-tubulin (GT114, GeneTex), polyclonal goat anti-rabbit-HRP (31460, Pierce), polyclonal goat anti-mouse-HRP (31430, Pierce). Following addition of HRP substrate (NEL113001EA, PerkinElmer), chemiluminescence was recorded with a LAS-400 mini Luminescent Image Analyzer and Image J was used for quantification.
8
Western Blot Analysis of Mitochondrial Proteins
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The whole cell lysates, cytosol fraction lysates, or mitochondrial lysates from healthy-hMSCs or CKD-hMSCs (30 μg protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in an 8–12% gel, and the proteins were transferred to a nitrocellulose membrane. After the blots were washed with TBST (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.05% Tween 20), the membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with the appropriate primary antibodies: against PrPC, MFN1, or β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), PINK1, OPA1, P62, LC3B, and VDAC1 (NOVUS, Littleton, CT, USA), and p-DPR1 (Cell signaling, Danvers, MA, USA). The membranes were then washed, and the primary antibodies were detected by means of goat anti-rabbit IgG or goat anti-mouse IgG antibodies (Santa Cruz Biotechnology). The bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).
9
Protein Extraction and Western Blot Analysis
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The protein of heart tissue and AC16 cells was extracted using whole Cell Lysis Assay Kit (KeyGEN Biotech, China) and then the concentration was detected by BCA kit (Dingguo Changsheng Biotech, China). The proteins were loaded in equal amounts, separated by SDS-PAGE gels and transferred to NC membranes. The membranes were incubated at 4 °C overnight with primary antibodies after blocking in TBS with skim milk (5%), which include ANP, PINK1 (NOVUS, USA); FTH1, Mfn2, LC3A/B, Drp1, Parkin, GAPDH (CST, USA); DHODH (Proteintech, USA); and BNP, FTMT, p62, COX-2, NCOA4, OPA1, 4-HNE, MDA (Abcam, UK). The next day, secondary antibody was used to incubate with the membranes. The ChemiDocTM Imaging System (Bio-Rad, USA) or the LI-COR Odyssey ® CLx Infrared Imaging System were employed to detect the signals of protein bands. Then, the Image Studio™ Software was used for visualization. Image J was used to analyze the density of the detected protein bands.
10
Mitochondrial Proteome Analysis in Frozen Tissue
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Frozen cortex tissue samples were lysed by mechanical homogenization with RIPA buffer containing protease and phosphatase inhibitors, and analyzed by SDS–PAGE and western blot. Subsequently, the concentration of extracted protein was determined by using the Bio-Rad Protein Assay. Proteins were detected using the following antibodies: HSP60 (Enzo Life Science), CLPP (Sigma), anti-GAPDH (14C10) (Cell Signaling), LONP1 (Sigma), PINK1 (Novus Biologicals), LC3 A/B (Cell Signaling), SDHB (Oxphos cocktail, Abcam), MTCO1 (Abcam), Ubiquitin (Enzo), P62 (BD Transduction Laboratories), Phopsho P62 (Cell Signaling), VDAC (Abcam). In addition to the housekeeping proteins, loading was monitored by Ponceau Red to ensure a homogeneous loading. Antibody detection reactions for all the immunoblot experiments were developed by enhanced chemiluminescence (Advansta) and imaged using the c300 imaging system (Azure Biosystems). Pixel intensity was quantified by using ImageJ software.
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