The cells or tissues were lysed with NP-40 lysis buffer (P0013F, Beyotime, Hangzhou, China) supplemented with 1 mM PMSF (TS505, Beyotime) as well as a protease inhibitor cocktail (Sigma-Aldrich). The protein concentration of samples was measured by the BCA assay (23227, Thermofisher). An equal amount of protein from the samples was subjected to SDS-PAGE and transferred to PVDF membranes (Millipore). Immunoblots were blocked with 5% defatted milk for 1 h and then incubated with the corresponding primary antibodies at 4°C overnight. After washing with TBST three times, membranes were incubated with the corresponding second antibodies for 2 h at room temperature. Finally, protein bands were visualized by a Pierce Chemiluminescence ECL kit (34577, Thermofisher), and the grayscale value was quantified by ImageJ software.
Pierce ecl chemiluminescence kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce ECL chemiluminescence kit is a product designed for the detection and quantification of proteins in Western blot analysis. It utilizes a chemiluminescent substrate to produce a luminescent signal proportional to the amount of target protein present in the sample. The kit provides the necessary reagents for efficient protein detection and can be used with a variety of imaging systems.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Np 40 lysis buffer, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase linked goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Antibodies for β catenin, by Merck Group (1 mentions)
Nuclear extraction kit, by Merck Group (1 mentions)
Calcineurin, by Cell Signaling Technology (1 mentions)
Lamin b1, by Santa Cruz Biotechnology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Tbs t, by Thermo Fisher Scientific (1 mentions)
Anti oligomer a11 antibody, by Thermo Fisher Scientific (1 mentions)
Syn1 antibody, by BD (1 mentions)
9 protocols using pierce ecl chemiluminescence kit
1
Western Blot Analysis of Protein Samples
2
Protein Expression Analysis via Western Blot
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To perform western blot analysis, the cells were treated with cell lysis buffer (CST, 9803) and inhibitor “cocktail” (Sigma, P8340). Protein concentration was measured by BCA assay (Pierce, 23235). An equal amount of protein was extracted from the sample for dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was electrically transferred to the polyvinylidene difluoride membrane (Bio-Rad). Immunoblots were probed with the indicated antibodies. The protein bands were visualized by Pierce Chemiluminescence ECL Kit (Thermo).
3
Immunoprecipitation and Western Blotting Protocol
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For immunoprecipitation, whole-cell extracts were lysed using IP Lysis Buffer (Pierce, 87785) supplemented with a protease inhibitor “cocktail” (Sigma, P8340). Cell lysates were centrifuged for 15 min at 12,000 g under 4°C, supernatants were collected and incubated with protein A/G magnetic beads (MedChemExpress, MCE, HY-K0202) together with specific antibodies. After overnight incubation, protein A/G magnetic beads were washed three times with IP wash buffer. Immunoprecipitates were eluted by SDS–PAGE loading buffer or Elution buffer (Pierce, 88848). For immunoblot analysis, cells were washed two times with cold PBS and lysed with cell lysis buffer (Cell Signaling Technology, 9803) supplemented with a protease inhibitor “cocktail” (Sigma, P8340). Protein concentrations of the extracts were measured by BCA assay (Pierce, 23235). Equal amounts of the extracts were loaded and subjected to SDS-PAGE, transferred onto polyvinylidene fluoride membrane (Millipore, IPVH00010), and then blotted as described previously (28 (link)). The specific protein bands were visualized by using a Pierce chemiluminescence ECL kit (Thermo Fisher Scientific, Waltham, MA, United States).
4
Protein Lysate Preparation and Western Blot Analysis
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Whole protein lysates from all tissues were prepared as previously described.41 (link) Whole protein lysates from cell lines were prepared by using RIPA lysis buffer (Santa Cruz Biotechnology Inc., Dallas, Texas, USA) according to the manufacturer’s instructions. Nuclear proteins were purified using a nuclear extraction kit (EMD Millipore, Billerica, MA, USA). SDS-polyacrylamide gel electrophoresis and western blot analyses were performed as previously described.33 (link) Antibodies for nuclear factor of activated T cells c1 (1:1 000) and lamin B1 (1:1 000) were from Santa Cruz Biotechnology. Antibody to detect irisin (epitope 42–112, 1:500) and FNDC5 expression was purchased from Phoenix Pharmaceuticals Inc. (#067-17). Antibodies for β-catenin (1:10 000) were from Sigma and those from P-AKT-1 (1:1 000), calcineurin (1:1 000), P-JNK (1:1 000), and β-actin (1:1 000) were from Cell Signaling (Danvers, MA, USA). The secondary antibodies were horseradish peroxidase-linked goat-anti-rabbit IgG (Santa Cruz Biotechnology). Blots were visualized using Pierce ECL chemiluminescence kit (Thermo Fisher Scientific).
5
Dot Blot Assay for α-Synuclein Oligomers
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Dot Blot assay with recombinant α-syn oligomers was performed as previously described [25 (link)]. Briefly, 0.2 – 2 μg α-syn oligomers were applied to a nitrocellulose membrane (pore size 0.22 μm, Whatman Protran, Sanford, ME, USA), blocked with 10 (w/v)% non-fat milkin Tris-buffered saline (TBS) containing 0.01 (v/v) % Tween 20 (TBS-T). The membrane was blocked with 10% (w/v) non-fat dried milk in TBS-T (Sigma-Aldrich, St. Louis, MO, USA) at RT for 1 h. After three washes with TBS-T, the membrane was incubated with anti-oligomer antibody A11 (1:100; Invitrogen, Carlsbad, CA, USA) or Syn-1 antibody (1:1000; BD transduction, Franklin Lakes, NJ, USA) overnight at 4 C with gentle agitation. The membranes were then washed three times for 5 min with TBS-T, incubated with horseradish peroxidase conjugated anti-rabbit IgG or anti mouse IgG (1:2000; Jackson Immuno Research Laboratories, Baltimore, PA, USA) in 5 (w/v) % non-fat dried milk in TBS-T and incubated for 1 h at RT. The blots were washed three times with TBS-T and developed with Pierce ECL chemiluminescence kit from Thermo Scientific (Rockford, IL, USA).
6
Western Blot Analysis of LC3B
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Collected cell pellets were extracted in RIPA buffer (20 mM HEPES, pH 7.0, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 10 mM β-Glycerophosphate, 1 mM Na3VO4, and 5 mM NaF) and protein concentrations were determined with the BCA protein assay kit (Thermo Fisher Scientific, Darmstadt, Germany; #23227). The proteins were separated on 12% gels by electrophoresis and were transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA; ISEQ00010). The membranes were blocked in 0.5% BSA in Tris-buffered saline with Tween-20 (TBS-T) and were probed with: anti-LC3B (Cell signaling, Danvers, MA, USA; #3868) and anti-α-tubulin (Sigma-Aldrich, Saint Louis, MO, USA; T5168), followed by secondary HRP-antibodies; anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA; sc-2005), and anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA; sc-2004). The blocking and probing were performed using SNAP i.d. 2.0 Protein Detection System (Merck Millipore, Billerica, MA, USA). Proteins were detected using Pierce ECL chemiluminescence kit (Thermo Fisher Scientific, Darmstadt, Germany; #32132). The bands were quantified by ImageJ (National Institutes of Health, Bethesda, MD, USA).
7
Comprehensive Liquid Chromatography-Mass Spectrometry Protocol
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All reagents for liquid chromatography (LC) and mass spectrometry (MS) analysis were of LC-MS grade and purchased from Merck (Darmstadt, Germany). Trypsin Gold was purchased from Promega (Madison, WI, USA). C18 solid phase extraction columns were purchased from Waters Corporation (Milford, MA, USA). Millipore Protease Inhibitor Cocktail Set I was purchased from Merck (Darmstadt, Germany). Invitrogen SuperScript™ IV First-Strand Synthesis System and Applied Biosystems PowerUp SYBR Green PCR Master Mix were purchased from Thermo Fisher Scientific (Vilnius, Lithuania). Ambion TRIzol Reagent was purchased from Sigma-Aldrich (St. Louis, MO, USA). Invitrogen nucleophosmin monoclonal antibody (NA24), Invitrogen natriuretic peptides A polyclonal antibody, Pierce BCA Protein Assay Kit and Pierce ECL chemiluminescence kit were purchased from Thermo Scientific (Rockford, IL, USA). The moesin polyclonal antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). The S100A8/A9 inhibitor (ABR-238901) was a gift from Active Biotech AB (Lund, Sweden).
8
Dot Blot Analysis of α-Synuclein
9
Native and Denatured Protein Detection
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