Cardiomyocytes were washed with PBS and lysed with a radioimmunoprecipitation assay (RIPA) lysis buffer(Beyotime biotechnology, China ) containing protease and phosphatase inhibitors (Sigma, U.S.A ).Then the protein was quanti ed by BCA assay (Thermo sher USA). Primary antibodies used included Anti-PINK1 antibody (1:1000, Abcam, USA), Anti-PGC-1α antibody (1:1000, Abcam, U.S.A) and anti-β-actin antibody (1:5000, Proteintech, USA). Second antibody used was goal anti-rabbit IgG-HRP (1:5000,Proteintech, USA). The protein bands were examined by ECL Substrate (FDbio, Hangzhou, China) and visualized by Gene Gnome Imaging System (Syngene Bio Imaging) and quanti ed by densitometry with Image J software (NIH).
Genegnome imaging system
Manufactured by Syngene
Sourced in United Kingdom, China, Germany, United States
The GeneGnome is a compact, high-performance imaging system designed for capturing high-quality images of nucleic acid gels and blots. It utilizes a sensitive cooled CCD camera and various illumination options to produce detailed, accurate results for applications such as DNA, RNA, and protein gel analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bca assay, by Thermo Fisher Scientific (4 mentions)
Genesnap software, by Syngene (4 mentions)
Sds page, by Bio-Rad (3 mentions)
Rabbit monoclonal antibodies, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ecl substrate, by Fdbio (2 mentions)
Anti pink1 antibody, by Abcam (2 mentions)
Anti β actin antibody, by Proteintech (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Anti β mhc, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (2 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Anti pgc 1α antibody, by Abcam (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
23 protocols using genegnome imaging system
1
Western Blot Analysis of Cardiomyocytes
2
Western Blot Analysis of Phosphorylated Kinases
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Equal amounts of sample protein (12 µg) were separated on 10% Precise SDS–PAGE gels, semi-dry transferred to nitrocellulose membranes and stained with Ponceau S to confirm homogeneous transfer. After blocking for 1 h in 5% non-fat dried milk, membranes were washed three times in 0.1% Tween-Tris-buffered saline (TTBS) and incubated in anti-phospho PKC (pan) (ζ Thr410), anti-phospho PKC (pan) (βII Ser660) or anti-phospho p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) antibodies (1∶1000 in TTBS) overnight at 4°C. After TTBS wash, blots were incubated for 2 h at room temperature with HRP-conjugated secondary antibodies (1∶3000 in TTBS) and exposed to West Pico chemiluminescence substrate. Immunoreactive bands were visualized with a GeneGnome imaging system (Syngene) and relative band intensities quantified using GeneTools software. Protein loading was determined by incubating blots with anti-actin antibodies (1∶1000). When necessary blots were stripped with Restore Western blot stripping buffer and incubated in an alternative antibody. In addition, to confirm that the anti-phospho PKC and anti-phospho ERK antibodies detected only the phosphorylated kinases, blots were treated with lambda phosphatase (400 U/ml in TTBS containing 1% BSA and 2 mM MnCl2) for 4 h before incubation in primary antibodies; secondary antibody labeling and detection were then performed.
3
Western Blot Protein Detection Protocol
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Briefly, a pre-wet nitrocellulose membrane immersed in Tris-buffered saline (TBS-T; 0.01M Tris-HCl at pH 7.5, 0.1 M NaCl, 0.05% (v/v) Tween-20) was loaded with 5 µl sample, standardised by particles/ml or 1 µg of cell lysate protein measured using the Pierce BCA Protein Assay Kit [Thermo Fisher Scientific (Life Technologies) Paisley, Scotland, UK] according to manufacturer’s instructions. Nonspecific sites were blocked by incubating the membrane in TBS+5% dried milk powder for 30 min. After washing twice in TBS-T, the membrane was incubated with primary antibody overnight at 4°C and secondary antibody for 1 h at RT, with TBS-T washes between each step. The membrane was developed using ECL reagents A and B and imaged using the Syngene GeneGnome imaging system and GeneSys software. A panel of antibodies used is shown in Supplementary Table S1
4
Western Blot Analysis of Cellular Proteins
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Total cellular proteins were extracted from dMϕ or decidual cells using TRIzol (Invitrogen, USA) or SDS lysis buffer (2% SDS, 50 mM Tris-HCI, pH 7.6, 2 mM EDTA, and 10% glycerol). The protein concentrations were quantified by using the BCA Protein Assay Kit (Thermo Pierce, USA). Then, 20 μg of protein was subjected to 10% SDS-PAGE and electrophoretically transferred to a PVDF membrane (Thermo Pierce). After blocking with 5% BSA, the membrane was incubated with different primary antibodies (Supplementary Table S3
5
Protein Expression Analysis in Cancer Cells
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Western blot analysis was used to detect the expression of total proteins and their phosphorylated forms in prostate cancer and bone cells. Briefly, cells were incubated in serum free medium with or without test agents for 60 min and then cells were treated with cytokines or conditioned medium from cancer cells and homogenized and collected in lysis buffer (0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1% Triton X-100, 1 mMEDTA, 2% (v/v) protease inhibitor cocktail, 10 mM of sodium fluoride and 2% (v/v) phosphatase inhibitor cocktail). Protein concentration was determined using BCA assay (Pierce, USA). Total protein (50-70 µg) was resolved by SDS-PAGE (BioRAD, UK) and immunoblotted and native and phosphorylated proteins were detected by using the indicated rabbit monoclonal antibodies (all at 1:1000 dilution), and immuno-complexes were visualized using chemiluminescence (Amersham, UK) on a Syngene GeneGnome imaging system. Bands were quantified using GeneSnap software (Syngene, UK) and level of actin (Sigma-Aldrich, UK) was used for normalization.
6
Western Blotting of Protein Samples
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Protein samples were loaded onto 4–12% (w/v) BisTris pre-cast gels (Invitrogen). Proteins were transferred to polyvinylidine fluoride (PVDF) membranes (Millipore), rinsed briefly in Tris-buffered saline plus 0.1% (v/v) Tween 20 (TBS-T), and blocked by incubation in TBS-T containing 5% (w/v) non-fat dry milk (blocking buffer) for 30 min. Primary antibody incubations were performed in blocking buffer overnight at 4 °C. Antibody concentrations used were: Anti-myc (Sigma), 1:2000 dilution; anti-FLAG (Sigma), 1:2000 dilution; anti-β-catenin (Cell Signaling Technologies), 1:2000; anti-Calnexin (AbCam), 1:3000 dilution; rabbit anti-LRRK2 (Epitomics ‘MJFF2’), 1:1000; and mouse anti-LRRK2 (NeuroMab ‘N138/6’), 1:1000. The following morning, membranes were washed three times in TBS-T, prior to incubation in HRP-conjugated secondary antibody (Santa Cruz) at 1:2000 in blocking buffer for 1 h at room temperature. After an additional three washes, staining was visualized using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Chemiluminescent Substrate (both Pierce) and a Syngene GeneGnome Imaging system. Images were quantified using GeneQuant software.
7
Western Blot Analysis of Cell Lysates
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The cells were washed three times with cold PBS and mixed with whole-cell lysis buffer (4 mM EGTA, 3 mM EDTA pH 8.0, 125 mM NaF, 0.5 mM NA 3 VO 4 , 2.5 mg/ml aprotinin, 25 mg/ml trypsin inhibitor, 12.5 mM HEPES pH 7.4, 1% Triton X-100, and 25 mM phenylmethylsulphonyl fluoride) as described previously (Zhang et al. 2013) (link). A BCA Protein Assay Kit (Pierce Biotechnology, Thermo Scientific, Dubuque, IA, USA) was used to determine the protein concentration by spectrophotometry at 562 nm (Beckman DU530, Fullerton, CA, USA). Twenty-five micrograms of each protein sample was subjected to western blotting analysis with the following primary antibodies: rabbit polyclonal anti-nephrin (1:500; ab58968, Abcam, Cambridge, MA, USA), rabbit polyclonal anti-human b-hCG (1:1000; ab54410, Abcam), mouse monoclonal anti-E-cadherin (1:1000; sc71008, Santa Cruz Biotechnology), mouse monoclonal anti-bactin (1:2000; TA-09, Zhongshan Golden Bridge Crop., Beijing, China), rabbit polyclonal anti-syncytin 2 (1:500; AP13018A, Abgent, San Diego, CA, USA), mouse monoclonal anti-connexin 43 (1:1000; 610061, Transduction Laboratories, Lexington, KY, USA), and HRP-conjugated secondary antibodies. Signals were detected by the GeneGnome Imaging System (Syngene Bio-imaging, Cambridge, UK).
8
Western Blot Analysis of PLAC8 Protein
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LoVo cells protein extracts were prepared using whole-cell lysis buffer (50 mM HEPES, 1 mM EGTA, 150 mM NaCl, 1.5 mM MgCl 2 , 100 mM NaF, 10 mM sodium pyrophosphate, 10% glycerol, and 1% Triton X-100) containing a protease inhibitor (Sigma-Aldrich). A total of 30 mg of protein was separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with 12% gel and transferred electrophoretically onto nitrocellulose blotting membranes (Pall Corporation). The membranes were blocked in 5% skim milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween 20) at room temperature for 1 hour. The membranes were incubated overnight at 4 C with specific primary antibodies against PLAC8 (A62753; Sigma-Aldrich) and b-actin (TA-09; Zhongshan Golden Bridge). Then the membranes were incubated with horseradish peroxidase conjugated secondary antibodies at room temperature for 1.5 hours. Immunodetection was performed with a Super Signal West Femto Trial Kit (Thermo Fisher Scientific), and images were acquired using the Gene Gnome Imaging System (Syngene Bio-imaging).
9
Quantitative Protein Analysis of Breast Cancer Cells
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Western blot analysis was used to detect protein expression in breast cancer cells. Cells were lysed in a standard buffer (0.1% (w/v) Sodium dodecyl sulfate, 0.5% (w/v) sodium deoxycholate, 1% Triton X-100, 1 μM Ethylenediaminetetraacetic acid, 2% (v/v) protease inhibitor cocktail, 10 µM of sodium fluoride and 2% (v/v) phosphatase inhibitor cocktail). Protein concentration was determined using BCA assay (Pierce, USA). Total protein lysate (60μg) was resolved by SDS-PAGE (BioRAD, United Kingdom), immunoblotted with antibodies according to manufacturer’s instructions, detected using rabbit monoclonal antibodies (all at 1:1000 dilution, cell Signalling Technology, USA) and immuno-complexes were visualised by enhanced chemiluminescence (Amersham, UK) on a Syngene GeneGnome imaging system. The intensity of the bands was quantified using GeneSnap software (Syngene, UK) and level of actin was used for normalization.
10
Protein Extraction and Western Blot Analysis
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Total protein was extracted from cells using RIPA buffer containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, 1 mM sodium fluoride, 1 mM sodium vanadate, 1% Triton X-100, 1% phosphatase and 1% protease inhibitors. The protein concentrations were quantified using the bicinchoninic acid method. Next, total protein (30 µg per lane) was subjected to 10% SDS-PAGE and transferred to PVDF membranes. Following transfer, membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 at room temperature for 2 h. After incubation with indicated primary antibodies [p-ERK (1:1,000), ERK (1:1,000), p-p38 (1:1,000), p38 (1:1,000), p-IκBα (1:1,000), IκBα, (1:1,000), p-JNK (1:1,000), JNK (1:1,000), p-p65 (1:1,000), p65 (1:1,000), NFATc1 (1:1,000), c-Fos (1:600), DC-STAMP (1:800), c-Jun (1:1,000), Lamin B1 (1:1,000) and GAPDH (1:1,000)] overnight at 4°C, the membranes were washed and then incubated with horseradish peroxidase-conjugated secondary antibodies diluted at 1:10,000 for 1 h at room temperature. ECL was used to develop a fluorescent signal. Antibody reactivity was detected using the Gene Gnome Imaging System (Syngene Europe) and band densities were quantified using ImageJ software 6.0 (National Institutes of Health). Only representative blots are shown.
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