Protein and mRNA expression was determined by western and northern blotting, respectively (7 (link),9 (link),15 (link)). Endothelial cells were lysed in electrophoresis buffer (125mM Tris [pH 6.8], 12.5% glycerol, 2% SDS, and trace bromophenol blue) and proteins separated by SDS-polyacrylamide gel electrophoresis. Following transfer to nitrocellulose membranes, blots were blocked with phosphate buffered saline (PBS) and non-fat milk (5%) and then incubated with antibodies against cyclin D1 (1:500), cyclin E (1:500), cyclin A (1:500), phospho-Rb (1:100), p27(1:300), p21 (1:500), cyclin B1 (1:500), cdk1, phospho-cdk1 (1:100), or β-actin (1:200). Membranes were washed in PBS, incubated with horseradish peroxidase-conjugated secondary antibodies and developed with commercial chemoluminescence reagents (Amersham, Arlington Heights, IL). For p21 mRNA expression, total RNA was loaded onto 1.2% agarose gels and fractionated by electrophoresis. RNA was blot transferred to Gene Screen Plus membranes and then incubated overnight at 68°C in hybridization buffer (Amersham, Arlington Heights, IL) containing [32P]DNA probes (1×108 cpm) for p21 or GAPDH. After hybridization, membranes were washed, and exposed to X-ray film. Protein and mRNA expression was quantified by scanning densitometry and normalized with respect to β-actin or GAPDH, respectively.
Hybridization buffer
Manufactured by Cytiva
Hybridization buffer is a solution used in molecular biology and genetics applications to facilitate the hybridization of nucleic acid molecules, such as DNA or RNA, to complementary sequences. It provides the necessary chemical conditions for the formation of stable and specific interactions between the target and probe sequences during hybridization experiments.
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2 protocols using hybridization buffer
1
Western and Northern Blot Analysis of Cell Cycle Regulators
2
Validation of Genomic Integration by Southern Blotting
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Proper integration of the pSN054-1437000-3xHA vector was confirmed by Southern blotting. Genomic DNA from the parent and edited lines was isolated (QIAamp DNA Blood Miniprep Kit), and 10 μg of each was digested with HinDIII, separated overnight on a 0.7% agarose gel and transferred to nylon (Nytran SuPerCharge TurboBlotter, 0.45 μm, GE Healthcare) overnight. The blot was then probed with the left homologous region (PCR product of 14APT-1/14APT-2) labelled with alkaline phosphatase (Amersham AlkPhos Direct Labeling Kit; GE Healthcare) in hybridization buffer (Amersham) at 55°C overnight, washed twice each in primary wash buffer (120 g/l urea, 1 g/l SDS, 100 ml/l 0.5 M sodium phosphate pH 7, 8.7 g/l NaCl, 2 g/l Amersham blocking reagent) and secondary wash buffer (6.05 g/l Tris base, 5.6 g/l NaCl, 2 ml/l 1 M MgCl2, pH 10), then detected with Amersham CDP-Star Detection Reagent (GE Healthcare) and exposed to blue autoradiography film (MidSci, BX810) overnight.
Additional validation by western blotting was done exactly as described in Polino et al. (2020) (link) (section ‘Validation of PMVAPT line’).
Additional validation by western blotting was done exactly as described in Polino et al. (2020) (link) (section ‘Validation of PMVAPT line’).
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