Gel doctm xr gel documentation system

The A549, NCI-H1299, and NCI-H460 cell lines were collected at 24 h following treatment or transfection and lysed in a protein extraction buffer for 30 min at 4°C. The Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology) was utilized to extract nuclear protein. The lysate concentrations were adjusted by measuring protein content according to bicinchoninic acid (BCA) method. Altogether 50 μg protein blended with 5× SDS loading buffer was added to 10%-15% SDS-PAGE gels. After first dimension separation, the protein was then transported onto the PVDF membranes. Later, 5% non-fat milk was used to block the membranes under ambient temperature and subsequently incubated using those above described primary antibodies under 4°C overnight, followed by 2 h of incubation using the HRP-labeled anti-mouse (1:500) or anti-rabbit (1:500) secondary antibody under room temperature. Later, the Gel DocTM XR⁺ Gel Documentation System (Bio-Rad, US) was utilized to detect bands using the enhanced chemiluminescence (ECL) reagents.

Genomic DNA was isolated from efficient bacterial isolates capable of growing in 1000 mg L^–1 of phenol and the diversity was analyzed using BOXA1R primer (5′-ACG GCA AGG CGA CGC TGA CG-3′) (Viswanath et al., 2015 (link)). Each 20 μl reaction containing 6 μl of 2X ampliqon red mix, 2 μl of 2.5 pmol primer, 2 μl of 50 ng template DNA was made up to 20 μl using double sterilized HPLC water. The PCR was carried by, initial denaturation at 94°C for 5 min, 35 cycles of 94°C for 3 s, 92°C for 30 s, annealing at 50°C for 1 min, extension at 68°C for 8 min, followed by final extension for 10 min then hold at 4°C. Around 20 μl of PCR products were electrophoresed for 6 h on 2% agarose gel prepared in 1X TAE buffer. The BOX-PCR DNA profiles were visualized under UV illumination and documented using a Gel Doc^TM XR+ Gel Documentation System (Bio-Rad, United States). The fingerprinting profiles were analyzed using GelJv.2 DNA tool by normalization, recognition, and assignment of bands on the gel by the Dice coefficient (Heras et al., 2015 (link)). The cluster analysis was performed by unweighted pair group method with arithmetic mean (UPGMA) algorithm and the dendrogram was constructed with similarity matrices.

Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with proteinase inhibitors. An equal amount of total protein for each sample was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) following conventional western blotting procedures as previously described (Bass et al., 2017 (link)). Primary antibodies against STK11 (1:1,000 (LS−C388827, Lifespan Biosciences, Seattle, USA)), β-actin (1:5,000) (NB100-56874, Novus, Littleton, USA), and horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000) (D110056, Sangon, Shanghai, China) were used. The protein bands were visualized using Gel DocTM XR+ Gel Documentation System (Bio-Rad, Hercules, CA). Band intensity was quantified using the ImageJ software.