Gel doctm xr

To confirm the apoptotic mode of cell death, DNA fragmentation assay was performed. MCF-7 and MDA-MB231 cells were treated with 50 and 100 µg/ml of BTB extract for 48 h. The lysis buffer (100 µl of 100 mM Tris pH-8.5, 5 M NaCl, 0.5 M EDTA, 0.05% TritonX-100, 10 µg/ml proteinase K and 10% SDS) was added to the pellet and incubated for 30 min on ice. The supernatant was collected in a fresh tube and mixed with 25:24:1 mixture of phenol: chloroform: isoamyl alcohol then precipitated with two equivalents of ice cold ethanol plus one-tenth equivalent of sodium acetate. This was followed by centrifugation at 12,000×g for 20 min. The pellet was re-suspended in 30 μl of sterile water–RNase solution (15 μg/ml RNase in sterile water) and subjected to electrophoresis in TE buffer (10 mM Tris-HCI, 1 mM EDTA, pH 8.0) on a 1% agarose gel and imaged in a Molecular Imager (Gel DocTM XR+) (BioRad, Hercules, USA).

Adipocytes, skeletal muscle cells or cell pellets were resuspended in RIPA buffer containing protease inhibitor cocktail and 1mM PMSF, sonicated on ice and lysates were centrifuged for 10 min at 10,000g and protein concentrations of supernatant were determined following a previously described method [10 ]. Protein from tissue extract or cell lysates or media was resolved on 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, Bedford, MA, USA) with the help of Wet/Tank Blotting System (Bio-Rad Laboratories Inc, Hercules, CA, USA). Membranes were probed with specific primary antibodies (1:1000) and then detected by using secondary antibody conjugated with alkaline phosphatise (1:3000). 5-bromro-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) was used for detection of the protein bands. Intensity of the bands was analyzed using Image Lab Software (Bio-Rad Gel DocTMXR+, USA). α-Tubulin was used as a loading control and it was not affected by incubation with insulin or dmp. Similarly, the amount of tubulin was not different among BL6, db/db and dmp treated db/db mice samples.

The gel mobility shift assay was performed as previously described with small modifications^{5 (link)}. In brief, an RNA molecular derived from nleA 5′UTR was synthesized with the sequence of 5′-CACUAAUAAUAUCAAUGGAUUGAUAUUAUUAAUG-3′ (Sangon Biotech). The RNA was dissolved in DEPC water, heated to 80 °C for 10 min, and slowly cooled down to room temperature before use. For the RNA-binding test, 1 μl pre-treated RNA solution (10 μM) was incubated, at room temperature for 30 min, with WT CsrA or the WT + C_del CsrA mixture or Re-CsrA at the indicated concentrations, and then resolved by 12% native TBE PAGE. For the experiments of CesT antagonizing the CsrA/RNA binding, decreasing concentrations of CesT were added to the pre-incubated CsrA/RNA (WT or WT + C_del) or Re-CsrA/RNA binding reactions and then further incubated for 2 h at room temperature. The resultant reactions were then resolved using 12% native TBE PAGE. In each case, the gel was stained with ethidium bromide and the RNA bands were visualized with Gel DocTM XR + (BIO-RAD).

Major chromosomal patterns were compared between WT and STM C. jejuni M1 DNA using PFGE. The RE SacII was chosen for DNA fragmentation as SalI, SmaI and KpnI (also used for PFGE of Campylobacter species)24 (link)25 (link)66 (link) all had recognition sequences within the Tn55 (link). The PFGE method used was based on those described in Rivoal et al. and Ribot et al.24 (link)25 (link). WT and STM C. jejuni M1 agar cultures, washed and diluted in PBS to an OD_600 nm of 0.6–0.8, were preserved and lysed within plugs made with 2% low-melt agarose (Promega) in TE pH 8. Genomic DNA within the agarose plugs was digested with SacII for 5 h at 21 °C, washed in 0.5x TBE and resolved by gel electrophoresis in 1% agarose in 0.5x TBE. Electrophoresis was performed according to manufacturer’s instructions at 6 V/cm for 20 h with a ramped pulse of 5–50 s using CHEF-DR^® II Pulsed Field Electrophoresis Systems (Bio-Rad) and connected to a LTD 20 cooling system (Hybaid). Gels were stained with 0.3x SYBR^® Safe (Invitrogen) and imaged on a GelDoc^TM XR + (Bio-Rad) with Image Lab 3.0 software (Bio-Rad).

Mycoplasma DNA was detected with a EZ-PCR Mycoplasma Detection Kit (Biological Industries, Israel) according to the manufacturer's instructions. The PCR products were then separated with 0.8% agarose gel electrophoresis and visualized under UV light (Gel DocTM XR, BIO-RAD, America).

DNA of A. flavus isolates was extracted as described in Lee et al. [89 (link)]. DNA was extracted by using a commercially available DNA extraction kit (DNeasy^® Plant Mini column kit, Qiagen, UK) by following the manufacturer’s instructions. The presence and quality of pure genomic DNA were confirmed through a 1% (w/v) agarose gel electrophoresis, and analysis was conducted at 110 V for 35 min. The gel was then viewed under a UV Transilluminator (Biorad Gel DocTM XR + Philadelphia, PA, USA) to confirm the presence of high molecular weight genomic DNA. The eluted DNA was stored at −80 °C until further molecular identification assays.

Genotyping of CRP harboring bla_NDM-1 was performed via PFGE. Chromosomal DNA was digested with 50U of SmaI (Takara, Dalian, China) for 3 h at 30 °C. Genomic DNA was separated by CHEF DRIII (Bio-Rad, Hercules, CA, USA) gel electrophoresis in a 1% agarose gel in 0.5 × TBE at 14 °C. The operating conditions are as follows: voltage, 6 V/cm; switch angle, 120°; switch time, 5–20 s for 19 h. XbaI-digested Salmonella Braenderup H9812 was used as the size ladder. The gels were stained with ethidium bromide (Macklin, Shanghai, China), digitally photographed with Gel Doc^TM XR+ (Bio-Rad, Hercules, CA, USA) and normalized as TIFF images. The DNA patterns were analyzed using BioNumerics software v7.6 (Applied Maths, Kortrijk, Belgium) to establish a dendrogram of strain relationships. UPGMA was used as a clustering algorithm and the Dice correlation coefficient was used with a 1.2% position tolerance. The strains with more than 85% similarity were considered to be related.