Iso-electric focusing (IEF) electrophoresis was performed using a vertical precast gel with pH range from 3 to 10 (SERVAGel IEF 3–10, SERVA Electrophoresis GmbH, Heidelberg, Germany). Samples were prepared according to the manufacturer’s protocol. Briefly, 2 µg of conjugated sdAb was mixed at least 1:1 with IEF sample buffer (SERVA Electrophoresis GmbH, Heidelberg, Germany) to a max volume of 35 µL, after which the samples were loaded in the gel. The inner chamber of the electrophoresis chamber was filled with IEF cathode buffer (SERVA Electrophoresis GmbH, Heidelberg, Germany), while the outer chamber was filled with IEF anode buffer (SERVA Electrophoresis GmbH, Heidelberg, Germany). Gels were run using following program: 1 h at 100 V, 2 h at 200 V, and finally, 30 min at 500 V. Afterwards, the gel was fixated in a 20% tricholoracetic acid (Acros Organics, Geel, Belgium) solution for 20 min, followed by staining in a crystal violet (SERVA Violet 17, SERVA Electrophoresis GmbH, Heidelberg, Germany)/20% phosphoric acid (Acros Organics, part of Thermo Fisher Scientific, Geel, Belgium) solution for 10 min. Then, the gel was destained in 3% phosphoric acid solution until a clear background was obtained. The gels were imaged using an Amersham 680RGB imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and analyzed via the GE ImageQuant TL 1D v 8.2.0 analysis software.
Amersham imager 680rgb
Manufactured by GE Healthcare
Sourced in Sweden, United States, Japan
The Amersham Imager 680RGB is a compact, high-resolution imaging system designed for life science research. It is capable of capturing fluorescent and chemiluminescent signals from a variety of samples, including gels, blots, and microplates. The system utilizes a high-sensitivity CCD camera and multiple illumination sources to provide efficient imaging across a range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Iblot2 transfer system, by Thermo Fisher Scientific (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Amersham ecl prime, by GE Healthcare (1 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Complete mini, by Roche (1 mentions)
Protease inhibitor cocktail, by CWBIO (1 mentions)
Peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Total protein extraction kit, by Invent Biotechnologies (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Imagej software, by Media Cybernetics (1 mentions)
Ab195377, by Abcam (1 mentions)
Ab194583, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Ab15277, by Abcam (1 mentions)
Ab129002, by Abcam (1 mentions)
Precision plus protein all blue standard, by Bio-Rad (1 mentions)
Criterion tgx precast gel, by Bio-Rad (1 mentions)
9 protocols using amersham imager 680rgb
1
Isoelectric Focusing of Single-domain Antibodies
2
SDS-PAGE Characterization of NOTA-sdAb
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SDS-PAGE was performed on NOVEX Wedgewell 16% 10-well gel (Thermo Fischer Scientific, Carlsbad, CA, USA), where 10 and 2 µg of NOTA-sdAb was loaded in both reducing and non-reducing conditions. The gel was run at 80 V for 10 min, then at 150 V for 65, after which a Coomassie Blue staining was performed for detection. Gels were visualized with the Amersham 680 RGB Imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and analyzed via the GE ImageQuant TL 1D v 8.2.0 analysis software.
3
Protein Extraction and Western Blot Analysis
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Cellular protein was extracted and analyzed as previously described [16 (link), 17 (link)]. Briefly, cells were washed three times with ice-cold PBS and lysed in lysis buffer containing 50 mM HEPES, 150 mM NaCl, 5 mM EDTA, 0.1% NP40, 20% glycerol, and protease inhibitor cocktail (Complete Mini, Roche, Indianapolis, IN) for 1 h. Cells were then transferred to a 1.5 ml tube and centrifuged. The supernatant was transferred into a new tube, and then the protein concentration was measured. Proteins (10 μg) were heated in SDS sample loading buffer at 70°C for 10 min and loaded onto NuPage 4–12% Bis-Tris gels (Thermo Fisher Scientific). After electrophoresis, proteins were transferred to a PVDF membrane using an iBlot 2 transfer system (Thermo Fisher Scientific). The membrane was washed with PBST (PBS with 0.1% Tween 20), blocked overnight with ImmunoBlock (KAC Co., Ltd., Kyoto, Japan), and then incubated with anti-GPAT3 (Thermo Fisher Scientific; 1:2,000 dilution) or anti-β-actin (Cell Signaling Technology, Danvers, MA; 1:2,000 dilution) antibodies. After washing with PBST, the membrane was incubated for 1 h with HRP- conjugated goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific; 1:1,500 dilution). The signal was developed using Amersham ECL Prime (GE Healthcare, Buckinghamshire, UK), and the images were scanned with an Amersham Imager 680 RGB (GE Healthcare).
4
Western Blot Analysis of Protein Expression
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Cells and heart tissues were collected, and lysates were prepared. An equal quantity of protein (40–60 µg) was resolved by SDS/PAGE and transferred to polyvinylidene difluoride membranes. The blots were blocked with 5% milk in Tris‐buffered saline with Tween 20 for 2 h at room temperature (RT) and then separately incubated with primary antibodies overnight at 4°C. The blots were then incubated with secondary antibodies conjugated to horseradish peroxidase for 1 h at RT and detected using a chemiluminescent instrument (GE, Amersham Imager 680RGB). Grayscale values for each blot were measured using the ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the intensity of each band was normalized to that of the loading control GAPDH or the total target protein. Detailed information regarding the antibodies used is provided in Table S3 (Supporting Information).
5
Western Blot Analysis of Protein Extracts
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Total protein was extracted from the left ventricular apex of mice using the Total Protein Extraction Kit (SD-001, Invent, USA). Protein lysates were extracted from cultured cells using RIPA lysis buffer (R0010, Solarbio, China) supplemented with a protease inhibitor cocktail (CWbio, China). Equal amounts of protein samples were separated on 10 to 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% skim milk for 1 hour at room temperature and incubated with primary antibodies at 4°C overnight. On the next day, the membranes were incubated with peroxidase-conjugated secondary antibodies (1:5000, Jackson ImmunoResearch Laboratories, USA) for 1 hour at room temperature, followed by detection using a chemiluminescent substrate (Millipore, USA) and chemiluminescent instrument (GE, Amersham Imager 680RGB, USA). Images were analyzed with ImageJ software (Media Cybernetics Inc., USA), and β-actin was used as a control to verify equal protein loading. The primary antibodies used were listed in table S1.
6
Protein Expression Analysis of Heart Tissues
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Samples comprising both heart tissues and cells were gathered, followed by the preparation of their lysates. Proteins, in consistent amounts ranging from 40 to 60 μg, were separated via SDS/PAGE and subsequently transferred onto PVDF membranes. These membranes were initially blocked using a 5% milk solution in Tris-buffered saline containing Tween 20 at ambient temperature for 2 h. This was followed by an overnight incubation at 4°C with primary antibodies (ALKBH5: 1:1,000, Abcam, Ab195377; Bcl-2: 1:2,000, Abcam, Ab194583; Bax: 1:10,000, Abcam, Ab32503, United Kingdom). Subsequently, the membranes were exposed to secondary antibodies (1:20,000; Proteintech, SA00001-2) linked to horseradish peroxidase for 1 h at room temperature, and detection was carried out using a chemiluminescence detection system (GE, Amersham Imager 680RGB, United States). The grayscale intensity of each blot was quantified using ImageJ software (provided by the National Institutes of Health, Bethesda, MD, USA), with band intensities normalized against the reference protein GAPDH (1:20,000; Proteintech, 10494-1-AP) or the aggregate target protein.
7
Membrane Protein Analysis of Cancer Cells
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The obtained cancer membrane (U87MG), GBM stem cell (X01) membrane, mitochondria membrane (MM), NPs, HM-NPs (U87MG) and HM-NPs (X01) were lysed with radio immunoprecipitation assay (RIPA) lysis buffer at 4 °C for 10 min. The lysates were subjected to SDS-PAGE and then transferred onto PVDF membranes (Millipore). After being blocked in 5% skim milk for 1 h, the membranes were separately incubated with rabbit antibodies against CD44, EpCAM, EHD2, Atlastin-1, and Mito-fusion, Integrin αv and Na+/K+ATPase at 4 °C overnight, respectively, and then treated with anti-rabbit secondary antibody (1: 10,000) for 1 h at room temperature. Subsequently, the immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Imager 680RGB, GE, Japan). Na+/K+ATPase was detected as a housekeeping protein control.
8
Gboxin-Induced Apoptosis Pathway in U87MG Cells
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U87MG cells were incubated in 6-well plates (2 × 105 cells/well) for 24 h, then added with 10 μL HM-NPs@G, CM-NPs@G, MM-NPs@G and free Gboxin (Gboxin: 800 nM), respectively. After 72 h incubation, the cells were washed three times with PBS and lysed with radio RIPA lysis buffer at 4 °C for 10 min. The lysates were subjected to SDS-PAGE and then transferred onto PVDF membranes (Millipore). After being blocked in 5% skim milk for 1 h, the membranes were separately incubated with rabbit antibodies against caspase-3/9 (C-3/9), cleaved caspase-3/9 (CC-3/9), cytochrome C (Cyto C) and β-actin at 4 °C overnight, respectively, and then treated with anti-rabbit secondary antibody (1: 10,000) for 1 h at room temperature. Subsequently, the immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Imager 680RGB, GE, Japan). β-actin was detected as a housekeeping protein control.
9
Dystrophin Protein Analysis by Western Blotting
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Proteins were analyzed by Western blotting. Each protein extract was mixed with one volume of Laemmli sample buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and boiled for 5 min. Precision Plus ProteinTM All Blue Standards (Bio-Rad Laboratories) were used as protein size markers. Equal amounts of protein (3 µg) were separated on 4-20% SDS-PAGE gels (Criterion TGX precast Gels, Bio-Rad) and electrotransferred to PVDF membrane using an iBlot2 transfer system (Thermo Fisher Scientific). The membranes were blocked with StartingBlock T20 blocking reagent (Thermo Fisher Scientific) and incubated overnight at 4 ℃ with 1:100 dilutions of antibodies to the N-terminal of dystrophin (NCL-DYSB, Leica Biosystems, Wetzler, Germany), the C-terminal of dystrophin (ab15277, abcam, Cambridge, UK) and a 1:1000 dilution of antibody to vinculin (ab129002, abcam) as a loading control. After washing, the membranes were incubated with a 1:20,000 dilution of secondary HRP-coupled antibody to rabbit for ab15277 and ab129002 (GE Healthcare Life Sciences) and to mouse for NCL-DYSB (GE Healthcare Life Sciences). After washing, the membranes were processed for enhanced chemiluminescence detection using Luminata Forte Western HRP substrate (EMD Millipore, Billerica, MA, USA). Immunoreactive proteins were visualized by Amersham Imager 680RGB (GE Healthcare Life Sciences).
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