Hippocampus and bacterial extracts were prepared in 0.5% CHAPS buffer containing complete protease inhibitor and centrifuged for 15 min at 20,000 g and 4°C, and protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Hippocampus and bacterial extraction supernatants, OMV and FBS samples were denatured in 6×Laemmli buffer, separated by SDS‐PAGE gel electrophoresis, and transferred to a nitrocellulose membrane. Following blocking with Odyssey blocking buffer (LI‐COR Biosciences), the membrane was incubated with primary antibodies, washed with PBS containing 0.1% Tween 20 and subsequently incubated with fluorophore‐ or HRP‐conjugated secondary antibodies. Protein bands were visualized by Odyssey Fc Imaging System (LI‐COR Biosciences) or WesternBright Quantum HRP substrate (advansta) in Amersham Imager 600 (GE Healthcare), and quantification was done in Image Studio (LI‐COR Biosciences) and Amersham Imager 600 integrated analysis software (GE Healthcare), respectively.
Amersham imager 600
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Japan, China, Sweden, Germany, Spain, France, Switzerland, Belgium, New Zealand
The Amersham Imager 600 is a compact and versatile imaging system designed for a wide range of life science applications. It features a high-resolution CCD camera and a selection of interchangeable emission filters to capture, analyze, and document fluorescent, chemiluminescent, and colorimetric signals.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (110 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (80 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (60 mentions)
Ripa buffer, by Thermo Fisher Scientific (55 mentions)
Bca protein assay kit, by Beyotime (39 mentions)
Polyvinylidene difluoride membrane, by Merck Group (37 mentions)
Pvdf membrane, by Bio-Rad (36 mentions)
Protease inhibitor cocktail, by Roche (34 mentions)
Trans blot turbo transfer system, by Bio-Rad (33 mentions)
Ripa buffer, by Beyotime (31 mentions)
Clarity western ecl substrate, by Bio-Rad (30 mentions)
Polyvinylidene fluoride membrane, by Merck Group (30 mentions)
Protease inhibitor cocktail, by Merck Group (29 mentions)
Ripa lysis buffer, by Beyotime (28 mentions)
β actin, by Merck Group (24 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (23 mentions)
Ripa buffer, by Merck Group (23 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (21 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (21 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (20 mentions)
1 560 protocols using amersham imager 600
1
Hippocampus and Bacterial Protein Analysis
2
CETSA Protocol for Cellular Target Engagement
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For CETSA, PC-3M, LNCaP-abl, PANC-1, and HepG2 cells were incubated with 1 µM of KMI169 or 1 µM KMI169Ctrl for 24 h. Then, cells were washed, harvested using trypsin, and pelleted by centrifugation. The pellet was washed twice with PBS supplemented with cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Roche) and Phosphatase Inhibitor Cocktail 2 and 3 (Sigma) and divided into 16 aliquots of 40 µl containing 150,000 cells (PC-3M), 145,000 cells (LNCaP-abl), 160,000 cells (PANC-1), or 200,000 cells (HepG2). Cells were then frozen in liquid nitrogen and thawed at 25 °C three times and each aliquot was heated to a defined temperature by applying a gradient between 40 °C and 70 °C in a PCR cycler (Mastercycler Nexus Gradient, Eppendorf; 16-well gradient, 55 °C ± 15). After 3 min incubation, aliquots were snap frozen in liquid nitrogen and thawed at 25 °C. Cell lysates were centrifuged at 20000x g (4 °C) for 20 min. Supernatants were mixed with 5x loading buffer, heated to 95 °C for 5 min, and analysed by Western blotting. Chemoluminescent signals were recorded with an Amersham imager 600 (GE Healthcare) and quantified using Amersham imager 600 1.2.0 software. Calculation of ΔTm was conducted using sigmoidal model in GraphPad Prism 6.0.
3
SDS-PAGE and BN-PAGE Analysis of Cytb6f Protein
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For SDS–PAGE analysis of purified cytb6f, protein samples were mixed with an equal volume of 2× Laemmli sample buffer (Merck) and boiled for 10 min prior to separation on precast NuPAGE 12% Bis-Tris gels (Invitrogen). For BN-PAGE analysis, cytb6f was diluted in 4× sample buffer (100 mM Tris–HCl pH 7.5, 0.05% (w/v) bromphenol blue, 40% (w/v) glycerol) and analyzed on precast NativePAGE 3–12% Bis-Tris gels (Invitrogen). Gels were stained with Coomassie Brilliant Blue and imaged using an Amersham 600 imager (GE Healthcare). Alternatively, SDS–PAGE separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (ThermoFisher Scientific) as described previously [37 (link)] and cytochrome c-mediated chemiluminescence was detected using the WESTAR ETA C 2.0 chemiluminescent substrate (Cyanagen) and an Amersham Imager 600 (GE Healthcare).
4
Protein extraction and immunoblotting of JAK-STAT pathway
5
Apoptosis Markers in Renal Tissues
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Protein expression levels of Bax, Bcl-2, and cleaved caspase 3 (Wanlei Biotechnology, Shenyang, China) in renal tissues were determined by western blot analysis. Briefly, frozen kidney tissues were lysed with radioimmunoprecipitation assay lysis buffer mixed with phenylmethylsulfonyl fluoride (Beyotime Biotechnology, Shanghai, China). After detection of total protein concentrations with a bicinchoninic acid assay kit (Beyotime Biotechnology), samples with equal volumes of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, which were incubated with primary antibodies of Bax (1 : 1000), Bcl-2 (1 : 500), and cleaved caspase 3 (1 : 1000) in Primary Antibody Dilution Buffer (Leagene Biotechnology, Beijing, China) overnight at 4°C. After washing, membranes were incubated with goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China) at 37°C for 2 h. All protein bands were captured with Amersham Imager 600 software (GE, Boston, MA, USA) and quantified with ImageJ.
6
Western Blot Analysis of Protein Expression
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Cell and lung samples were homogenized in RIPA buffer containing protease and phosphatase inhibitor cocktails. The total protein content in the tissue lysates was quantified using the BCA protein assay kit. Equal amounts of protein samples were separated by SDS-PAGE. Proteins were, then, transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). The membranes were incubated overnight at 4 °C with primary antibodies diluted in TBST (TBS containing 0.1% Tween-20) buffer containing 3% non-fat milk, followed by incubation with corresponding secondary antibodies for 1 h at room temperature. Immunoreactive bands were detected using the enhanced ECL chemiluminescence reagent. The membrane was stripped using the Western blot stripping buffer and subsequently labeled with β-actin antibodies following the procedure described above. Immunoreactive bands were captured using the Amersham Imager 600 software (GE Healthcare).
7
Analyzing Apoptosis Signaling Pathways
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Hippocampal tissue and PC12 cells were lysed in RIPA buffer with PMSF and phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China). After the protein concentration was determined using the BCA assay kit, the equivalent amount of protein samples was separated by SDS-PAGE gel electrophoresis, and transferred to the PVDF membrane. Then, membranes were blocked in 5% skim milk for 2 h at room temperature. Subsequently, membranes were incubated with primary antibodies adding Primary Antibody Dilution Buffer (Leagene Biotechnology, Beijing, China) overnight at 4℃. The primary antibodies include Bax, cleaved caspase-3, cleaved caspase-9 (1:1000, Cell Signaling Technology, USA); Bcl-2, cytochrome C, P-JNK, JNK, P-ERK, ERK, P-p38, p38 (1:750, Wanlei, Shenyang, China); c-Myc, CLIC4
(1:1000, Santa Cruz Biotechnology Inc, USA). Next, they were incubated with appropriate combination of secondary antibody for 2 h at 37°C. The immunoreactive protein bands were visualized using the enhanced chemiluminescence kit (Beyotime Biotechnology, Shanghai, China), and were captured by Amersham Imager 600 software (GE, USA). Finally, all protein bands were quanti ed with ImageJ software.
(1:1000, Santa Cruz Biotechnology Inc, USA). Next, they were incubated with appropriate combination of secondary antibody for 2 h at 37°C. The immunoreactive protein bands were visualized using the enhanced chemiluminescence kit (Beyotime Biotechnology, Shanghai, China), and were captured by Amersham Imager 600 software (GE, USA). Finally, all protein bands were quanti ed with ImageJ software.
8
Analyzing Apoptosis Signaling Pathways
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Hippocampal tissue and PC12 cells were lysed in RIPA buffer with PMSF and phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China). After the protein concentration was determined using the BCA assay kit, the equivalent amount of protein samples was separated by SDS-PAGE gel electrophoresis, and transferred to the PVDF membrane. Then, membranes were blocked in 5% skim milk for 2 h at room temperature. Subsequently, membranes were incubated with primary antibodies adding Primary Antibody Dilution Buffer (Leagene Biotechnology, Beijing, China) overnight at 4℃. The primary antibodies include Bax, cleaved caspase-3, cleaved caspase-9 (1:1000, Cell Signaling Technology, USA); Bcl-2, cytochrome C, P-JNK, JNK, P-ERK, ERK, P-p38, p38 (1:750, Wanlei, Shenyang, China); c-Myc, CLIC4
(1:1000, Santa Cruz Biotechnology Inc, USA). Next, they were incubated with appropriate combination of secondary antibody for 2 h at 37°C. The immunoreactive protein bands were visualized using the enhanced chemiluminescence kit (Beyotime Biotechnology, Shanghai, China), and were captured by Amersham Imager 600 software (GE, USA). Finally, all protein bands were quanti ed with ImageJ software.
(1:1000, Santa Cruz Biotechnology Inc, USA). Next, they were incubated with appropriate combination of secondary antibody for 2 h at 37°C. The immunoreactive protein bands were visualized using the enhanced chemiluminescence kit (Beyotime Biotechnology, Shanghai, China), and were captured by Amersham Imager 600 software (GE, USA). Finally, all protein bands were quanti ed with ImageJ software.
9
Western Blotting for Protein Analysis
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Cell lysates were processed with 6× Laemmli sample buffer, followed by electrophoresis. Samples were transferred to polyvinylidene difluoride membranes and blocked with 5% milk in TBST (Sigma-Aldrich, St. Louis, MO, USA). Western blotting was performed with the following antibodies at 4 °C overnight: anti-pMAP3K9 (T312) (cat. no.: BS-6779R, Thermo Fisher Scientific, Waltham, MA, USA), anti-MAP3K9 (cat. no.: ab228752, abcam, Cambridge, UK), anti-p70 S6K, anti-p-Akt (S473), anti-Akt, anti-pMEK1/2(S217/221) (cat. no.: 9204S, 9271S, 9272, 9121S; Cell Signaling Technology, Inc., Danvers, MA, USA), and anti-GAPDH (cat. no.: GTX100118, GeneTex, Irvine, CA, USA). Membranes were washed with TBST twice and incubated with a horseradish peroxidase (HRP)-antibody at room temperature for 1 h. After incubation with an HRP substrate (WesternBright ECL HRP Substrate, Advansta, San Jose, CA, USA), images were taken with an AmershamTM Imager 600 (GE Healthcare, Chicago, IL, USA).
10
HINT1 Binding Interactions with Adenine Nucleotides
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HINT1WT (25 μM) or HINT1H114A were mixed with either 700 μM Ap3A, Ap4A, Ap5A, AMP, ATP, or blank buffer and incubated at 4 °C rotating gently for 2 h. The working buffer includes 25 mM HEPES pH 8.0, 400 mM NaCl, and 2 mM EDTA. Samples were separated by 4–20% gradient SDS-polyacrylamide gel electrophoresis (PAGE) without pre-heating. RealBand 3-color High Range Protein Marker (BBI Life Science, Rockville, MD) was used to identify molecule weight of target bands. Gel under 25 kDa marker was cut for Coomassie brilliant blue staining. Gel above 25 kDa was transferred onto nitrocellulose membranes. Blottings were blocked in 5% non-fat milk for 30 min at room temperature, then incubated in Anti-His antibody (TransGen Biotech, catalog number HT501-02, dilution 1:3000) overnight at 4 °C and follow by 1 h incubation in horseradish peroxidase (HRP)-conjugated Goat Anti-Mouse IgG (BBI Life Science, catalog number D110087, 1:5000 dilution) at room temperature. Blottings were then treated with Pierce ECL Western Blotting Substrate and visualized with Amersham TM Imager 600 (GE Healthcare, Piscataway, NJ). The molecular weights of protein bands were calculated with three independent experiments by Amersham TM Imager 600 Analysis Software Version1.0 (GE Healthcare, Piscataway, NJ).
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