Steady-state levels of pre-rRNAs were assayed using northern blot and primer extension anaysis47 (link). Ten milliliter of cells were harvested, frozen, and RNA was extracted using phenol. Five microgram of RNA was used for primer extension reactions or loaded onto a formaldehyde/MOPS agarose gel for northern blotting. 32Pγ -ATP radiolabeled oligonucleotide probes for specific pre-rRNAs were used in primer extension reactions and for hybridization in northern blots. For northern hybridization of small molecular weight RNAs (7, 6, 5.8, and 5S), RNA samples were mixed with an equal volume of sample buffer (0.1× TBE buffer, 10 M urea, 0.1% xylene cyanol, 0.1% bromophenol blue) and subjected to electrophoresis on a 5% acrylamide/7 M urea gel for 4 h at 120 mA. Following electrophoresis, gels were electroblotted to a Nytran N membrane (GE Healthcare Life Sciences) using a Trans-Blot Plus Cell (Biorad), hybridized with an end-labeled oligonucleotide, washed, and exposed to X-ray film.
Trans blot plus cell
Manufactured by Bio-Rad
The Trans-Blot Plus Cell is a laboratory instrument used for the transfer of proteins from polyacrylamide gels to membranes. It provides a consistent and efficient method for protein blotting, enabling researchers to analyze protein expression and modifications.
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2 protocols using trans blot plus cell
1
Pre-rRNA Steady-State Levels Quantification
2
Identifying Conserved Immunogenic Proteins
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Enriched liposoluble proteins from PG2 T and from the 3 field isolates were pooled to identify conserved proteins that induce an immune response. Pools were resolved both by SDS-PAGE and 2D PAGE. Subsequently, proteins were transferred onto nitrocellulose membranes with a Mini-Trans-Blot Cell (Bio-Rad) at 250 mA for 1 hour (1D-WB), or with a Trans-Blot ® Plus Cell (Bio-Rad) at 0.15 A O/N at 4°C (2D-WB). After blotting, membranes were blocked with PBS-T containing 5% skim milk and incubated for 1 h with individual sera collected from naturally infected sheep, or pooled sera, in both cases diluted 1:1000 in PBS-T containing 2% skim milk. After 3 washes with PBS-T, membranes were incubated with anti-sheep IgG secondary HRP-conjugated antibodies (Southern Biotech) diluted 1:50.000 in PBS-T containing 2% skim milk. After 5 washing steps, membranes were developed with Chemiluminescent Peroxidase Substrate (Sigma-Aldrich) and images were acquired with a VersaDoc MP 4000 Imaging System (Bio Rad).
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