Total cell lysates were obtained from PC12 cells using radioimmunoprecipitation assay buffer solution (Beyotime Institute of Biotechnology) containing proteinase and phosphatase inhibitors (Beijing Sangon Biotech Co., Ltd.). The protein concentration was determined using a bicinchoninic acid protein assay kit (Sigma-Aldrich; Merck KGaA). The supernatant (30 µg protein) from the cell lysates was separated using SDS-PAGE (10%) and blotted on to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). The membranes were subsequently blocked with 5% non-fat milk for 1.5 h in Tris-buffered saline (TBS)/Tween 20 buffer at 25°C and then incubated overnight with the primary antibodies against caspase-3 (1:800), Bax (1:1,000), Bcl-2 (1:900), or β-actin (1:1,500; used as the control protein for normalization of the blots) at 4°C. After washing three times with TBS, the membranes were incubated at 25°C for 3 h with goat anti-mouse IgG-HRP (1:1,500; cat. no. sc-2005) or goat anti-rabbit IgG-HRP (1:1,500; cat. no. sc-2004); the second antibodies were purchased from Santa Cruz Biotechnology, Inc. Detection of the signal was performed using an enhanced chemiluminescence system (Pierce; Thermo Fisher Scientific, Inc.), and the intensity of the bands was determined via scanning and quantification using the Bio-Rad Gel Doc™ 2000 imaging system (Bio-Rad Laboratories, Inc.).
Gel doc 2000 imaging system
Manufactured by Bio-Rad
Sourced in United States
The Gel Doc 2000 imaging system is a versatile laboratory instrument designed for the capture and analysis of gel-based images. It utilizes a charge-coupled device (CCD) camera and specialized software to provide high-quality digital images of electrophoresis gels, blots, and other gel-based applications.
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Lab products found in correlation
Quantity one software, by Bio-Rad (3 mentions)
Bicinchoninic acid protein assay kit, by Merck Group (1 mentions)
Radioimmunoprecipitation assay buffer solution, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Sybr safe, by Thermo Fisher Scientific (1 mentions)
Reverse transcriptase, by Promega (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Cocktail of protease inhibitor, by Roche (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (1 mentions)
Amplitaq gold dna polymerase, by Thermo Fisher Scientific (1 mentions)
Fastgene gel pcr extraction kit, by Nippon Genetics (1 mentions)
Agarose gel, by Merck Group (1 mentions)
Ethidium bromide, by Bio-Rad (1 mentions)
Ecl plus kit, by Thermo Fisher Scientific (1 mentions)
Anti ulk1, by Cell Signaling Technology (1 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
27 protocols using gel doc 2000 imaging system
1
Protein Expression Analysis of Apoptotic Regulators in PC12 Cells
2
Inhibition of HsrA DNA Binding by DHP Derivatives
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The inhibitory action of DHP-based HHQ derivatives on the in vitro DNA binding activity of HsrA was assessed by electrophoretic mobility shift assay (EMSA), as previously described (González et al., 2019b (link)). Briefly, recombinant HsrA protein (5 μM) was mixed with 120 ng of its target promoter (PporGDAB) in a 20 μl reaction volume containing 10 mM bis-Tris (pH 7.5), 40 mM KCl, 100 mg/L BSA, 1 mM DTT and 5% glycerol, in the presence of 3, 2, and 1 mM of each 1,4-DHP derivative. DMSO instead of DHP was included as vehicle control, while an internal sequence of the gene pkn22 (alr2502) from Anabaena sp. PCC 7120 was used as non-specific competitor DNA in all assays. Mixtures were incubated at room temperature for 20 min and subsequently separated in a 6% native polyacrylamide gel electrophoresis. Gels were stained with SYBR Safe® (Thermo Fisher Scientific) and analyzed by using a Bio-Rad Gel Doc 2000 Imaging System (Bio-Rad Laboratories, Hercules, CA, United States).
3
Quantitative Real-Time PCR Analysis
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Total RNA was extracted using Trizol (Invitrogen-Life Technologies). Single-strand cDNA was synthesized with reverse transcriptase (Promega, Madison, WI, USA). The primers of Bax, Bcl-2, and β-actin are as follow: Bcl-2, forward 5′-TACCGTCGTGACTTCGCAGAGAT-3′ and reverse 5′-AGGAGAAATCAAACAGAGGTCGC-3′. Bax, forward 5′-TTTGCTACAGGGTTTCATCCAGG-3′ and reverse 5′-CAAAGTAGAAGAGGGCAACCACG-3′. β-actin, forward 5′-GTCCCTCACCCTCCCAAAAG-3′ and reverse 5′-GCTGCCTCAACACCTCAACCC-3′. Polymerase chain reaction (PCR) amplification was performed as follow: 94°C for 5 minutes followed by 40 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and an extension for 10 minutes at 72°C. The amplification products were separated on 1.5% agarose gel electrophoresis containing ethidium bromide, and examined using a Gel Doc 2000 Imaging System (Bio-Rad, Hercules, CA, USA).
4
Quantitative Analysis of Immunoblotting
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Quantitative analysis of immunoblotting was performed using Quantity-One software (Gel Doc 2000 imaging system, Bio-Rad Laboratories, Inc., Hercules, CA, USA). For protein level, the protein ratio of BLM (band density of protein/band density of β-actin) was set as 100% in the BPH or NC group. The data from other groups were expressed as a percentage of the BPH or NC group. The values are presented as the mean ± standard error. Statistical analysis was conducted by one-way analysis of variance followed by all pairwise multiple comparison procedures using the Bonferroni test and Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
5
Protein Extraction and Western Blot Analysis
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The RIPA buffer [20 mmol/L Tris-HCl (pH 7.4), 2 mmol/L EGTA, 150 mmol/L sodium chloride, 1% Triton-X 100, 0.5% sodium deoxycholate, 2 mmol/L sodium orthovanadate and 100 mmol/L sodium fluoride] containing a cocktail of protease inhibitors (Roche, West Sussex, United Kingdom) was used to extract proteins from rat hearts. The concentrations of the extracted proteins were quantified by PierceTM BCA Protein Assay Kit. Then the proteins were boiled for 5 min and subsequently separated by electrophoresis on a 12% SDS polyacrylamide gel. After separating, the proteins were transferred to PVDF membranes. The membranes were incubated with the primary antibodies against Bax, Bcl-2, Caspase-3, JAK2, p-JAK2, STAT3, p-STAT3, and β-actin overnight at 4°C. Then, the membranes were washed with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibody in TBST buffer at 37°C for 2 h. Finally, the membranes were washed and visualized using PierceTM ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, Cat No. 32132), and the bands were scanned and quantified using Bio-Rad Gel Doc 2000 imaging system.
6
Fluorescence Assay Instrumentation Protocol
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All fluorescence assay measurements were performed on an RF-5301PC system (Shimadzu, Japan). The apparatuses used to prepare the aptamer and H1 and H2 hairpins included a Mini-7k (Allsheng, Shanghai), an M-Microcentrifuge (AndyBio, Beijing), an XW-80A vortex mixer (Qilinbeier, Jiangsu) and a T100™ thermal cycler (Bio-Rad, USA). The instruments used for gel electrophoresis were a DYY-2C system (LIUYI, Beijing) and a DYCZ-24EN system (LIUYI, Beijing). Electrophoresis imaging was performed with a Bio-Rad gel Doc 2000 imaging system for gel analysis.
7
Genotyping of Floxed Alleles in Mice
8
Mycoplasma Neuraminidase Gene Sequencing
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PCR products from both PCR reactions were separated on a 1.8% agarose gel (Sigma-Aldrich, Burlington, MA, USA) at 130 V for 30 min. The gel was stained with ethidium bromide (BioRad, Hercules, CA, USA) and the results were visualized using BioRad Gel Doc 2000 Imaging System on Quantity One 4.4.0 Analysis Software. PCR amplicons from the second PCR (primer pair Ca-NEU-4F/Ca-NEU-4R) of appropriate length were excised from the agarose gel and purified using the FastGene Gel/PCR Extraction Kit (Nippon Genetics Europe, Düren, Germany) according to the manufacturer’s instructions. The purified PCR amplicons were Sanger sequenced at Macrogen Europe (Amsterdam, The Netherlands). Sequences were analyzed using Finch TV 1.4.0 (Geospiza Inc., Seattle, WA, USA) and queried against the GenBank database [30 (link)] using BLASTn [31 (link)]. Obtained partial neuraminidase gene sequences were deposited into the GenBank database. A Clustal Omega alignment [32 (link)] of partial neuraminidase gene sequences was carried out, comparing the nucleotide sequences to other known mycoplasma neuraminidase genes, accessible in the GenBank database.
9
Clonogenic Survival Assay of Irradiated Caco2 Cells
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Caco2 cell clones (control and KR4) were grown in 75 cm2 flask to reach 80% confluence. Then cells were trypsinized, counted and irradiated at different doses of γ-radiation in a Cs-137 irradiator. Cells were plated in 6 well plates in triplicates (EMEM with 10% FCS). After two weeks of incubation, colonies were fixed with 3:1 ratio of methanol-acetic acid and stained with 0.5% crystal violet solution. Colony images were taken on Gel doc-2000 imaging system and counted using Quantity One software (Bio-Rad laboratories). The surviving fraction was normalized to the corresponding un-irradiated control. For Rad51 inhibitor study, cells were treated with 27 μM of BO2 (Rad51 inhibitor) or DMSO (vehicle 1.35%) for 1 h and then the clonogenic assay was performed.
10
Native Agarose Gel Electrophoresis of NTA-DOX and FA-Conjugated HisHBcAg Nanoparticles
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The migration profiles of the HisHBcAg nanoparticles conjugated with NTA-DOX and FA were examined with NAGE. The samples were electrophoresed on a native agarose gel as described by Biabanikhankahdani et al.9 (link) and Yoon et al.41 (link). DOX bands were visualised by ultraviolet illumination (UV) using the GelDoc 2000 Imaging System (Bio-Rad, Philadelphia, PA, USA), while proteins were stained with CBB.
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