Cells were lysed with 200 μl of Lysis buffer (50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF). 18 μl samples were then loaded onto gels alongside 8 μl of protein ladder. The proteins were then transferred to nitrocellulose membranes before incubation in blocking buffer (5% (w/v) skimmed milk powder in TBS-T) for 1 hour at room temperature. The blots were then washed in TBS-T and incubated overnight with anti-NFATc2 (NFAT1) (BD Transduction Laboratories) or anti-phospho-p65 (S536) (Cell Signaling Technologies) primary antibodies (BD Transduction Laboratories) at 1:5000 and 1:1000 dilution respectively, in 5% (w/v) BSA in TBS-T. Blots were washed in TBS-T and then incubated for a further hour with a 1:2000 dilution of HRP-conjugated anti-IgG (Cell Signaling Technology). After a final TBS-T wash to remove any unbound antibody, a 1:1 ratio mix of ECL Advance Western Blotting Detection kit reagents was then applied to the membrane and left for 1 minute, before visualizing HRP-conjugated proteins using a Bio-Rad Gel Doc XRS+ System. Membranes were then re-stained with an anti-α-Tubulin primary antibody (Cell Signaling Technologies) and the detection process was repeated in order to produce a loading control.
Gel doc xrs system
Manufactured by Bio-Rad
Sourced in United States
The Gel Doc XRS+ System is a digital imaging system designed for capturing and analyzing images of DNA, RNA, and protein gels. It features a high-resolution camera, adjustable UV transilluminator, and comprehensive imaging and analysis software. The system is used for applications such as gel electrophoresis and western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho p65 s536, by Cell Signaling Technology (2 mentions)
Anti nfatc2 nfat1, by BD (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti α tubulin primary antibody, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti mouse igg, by Merck Group (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ha from streptococcus zooepidemicus, by Merck Group (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Kod dna polymerase, by Toyobo (1 mentions)
8 protocols using gel doc xrs system
1
Western Blot Analysis of NFAT and NF-κB Signaling
2
Protein Extraction and Western Blotting of Spinal Cord
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Total proteins were extracted from spinal cords by homogenization of tissue samples in a RIPA buffer (Sigma) containing protease and phosphatase inhibitors as previously described [12 (link)]. Protein concentrations were measured by Protein Assay BCA Kit (Thermo Scientific) with bovine serum albumin (BSA) as standard. Protein extracts (40 μg) were separated with 4%–13% gradient sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted to polyvinylidene fluoride membranes. After incubation with Krox 20, peripherin, P0, p75, and HRP-conjugated anti-mouse/anti-goat IgG (Sigma), blots were visualized with a mixture of 1.25 mM luminol, 146 mM p-coumaric acid, and 34% H2O2. The detection and analysis of immune complexes were performed by the Gel Doc XRS+ System (Bio-Rad, Hercules, CA, USA). Bands were quantified with the use of ImageJ version 1.46, and densitometric levels of proteins were normalized to β-actin.
3
Quantifying Spinal Cord GFAP Levels
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Total protein was isolated from spinal cords by homogenization of tissue samples in RIPA buffer (Sigma). Protein concentrations were determined using a Protein Assay BCA Kit (Thermo Scientific). Protein extracts (40 μg) were analyzed by 4%–13% gradient SDS-PAGE and then transferred to PVDF membranes. After incubation with glial fibrillary acidic protein (GFAP) (1:200, mouse monoclonal, Santa Cruz) and HRP-conjugated anti-mouse IgG (Sigma), blots were visualized with a mixture of 1,25mM luminol, 146mM p-coumaric acid and 34% H2O2. Detection and analysis of immune complexes was performed using the Gel Doc XRS+ System (Bio-Rad, Hercules, CA, USA). Bands were quantified using Image J Version 1.46, GFAP densitometric levels were normalized to β-actin. We also performed positive and negative controls using Western Blotting control for GFAP antibodies (sc-115582, Santa Cruz) and protein extracts from mononuclear umbilical cord blood cells (which does not contain GFAP), respectively.
4
DNA Quantification and Gel Analysis
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DNA quantification was carried out using the Nanodrop (ThermoScientific). DNA samples were run on a 1% electrophoresis gel and analyzed using a Gel Doc™ XRS System and Quantity One® software (Bio-Rad Laboratories).
5
Western Blot Analysis of NFAT and NF-kB Signaling
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Cells were lysed with 200 μl of lysis buffer (50 mM Tris-HCl [pH 7.4], 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF). Samples (18 μl) were then loaded onto gels alongside 8 μl of protein ladder. The proteins were then transferred to nitrocellulose membranes before incubation in blocking buffer (5% [w/v] skimmed milk powder in TBST) for 1 h at room temperature. The blots were then washed in TBST and incubated overnight with anti-NFATc2 (NFAT1) (BD Transduction Laboratories) or anti–phospho-p65 (S536) (Cell Signaling Technology) primary Abs (BD Transduction Laboratories) at 1:5000 and 1:1000 dilution, respectively, in 5% (w/v) BSA in TBST. Blots were washed in TBST and then incubated for a further hour with a 1:2000 dilution of HRP-conjugated anti-IgG (Cell Signaling Technology). After a final TBST wash to remove any unbound Ab, a 1:1 ratio mix of ECL Advance Western blotting detection kit reagents was then applied to the membrane and left for 1 min, before visualizing HRP-conjugated proteins using a Bio-Rad Gel Doc XRS+ system. Membranes were then restained with an anti–α-tubulin primary Ab (Cell Signaling Technology), and the detection process was repeated to produce a loading control.
6
HA Zymography for Hyaluronidase Activity
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Hyaluronidase activity was assayed via HA zymography as previously described (Monzon et al., 2010 (link)). Briefly, the samples were electrophoresed in 10% polyacrylamide gels containing 0.17 mg/ml HA from Streptococcus zooepidemicus (Sigma-Aldrich) followed by a 2-h incubation in 0.3% Triton X-100 at room temperature. The gels were subsequently incubated overnight in 0.15 M NaCl containing 0.1 M sodium formate, pH 3.7, at 37°C and then stained with 0.5% Alcian blue containing 3% acetic acid for 16 h. HYAL2 activity was visualized as clear bands on the blue background. Band intensities were recorded with a GelDoc XRS system (Bio-Rad) and analyzed using Quantity One software (Bio-Rad). The results were expressed as percentage change in intensity (INT) per square millimeter of surface.
7
Genomic DNA Extraction and Mutagenesis Analysis
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For each BAR-positive transgenic line, three individual tissue samples were used to extract genomic DNA. Maize genomic DNA was extracted with the hexadecyltrimethylammonium bromide method [23 (link)]. Target regions were amplified with specific primers pairs flanking the designed target sites (see Additional file 1 : Table S1 for primer sequences) using KOD DNA polymerase (Toyobo) to detect mutagenesis at the desired sites. The PCR product was separated on a 1 % agarose gel and stained using ethidium bromide. The stained gels were imaged using the Gel Doc XRS system (Bio-Rad). Selected PCR products were cloned into the pGEM-T Easy Vector (Promega) for DNA sequencing. For PCR product of each tissue sample, twenty clones were sequenced to detect stable editing.
8
Maize Transgene Detection via PCR
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The hexadecyltrimethylammonium bromide method was used for genomic DNA extraction in the maize plants [25] (link). Through PCR, we determined whether the transgenic plants were positive. The existence of pHB vectors was determined by amplifying HPT, and the pCA-GFP vector was directly and specifically amplified with specific primers targeting GFP (Table S2). In general, the gene was amplified with Taq mix in a PCR thermocycler. The PCR products were electrophoresed in a 1% agarose gel with ethidium bromide and imaged by the Gel Doc XRS system (Bio-Rad). The sizes and locations of specific gene bands were compared to determine the existence of the relevant genes.
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