α-变形杆菌核糖体的纯化及Northern印迹分析

Liang  Yin; Caroline S. Harwood

doi:10.21769/BioProtoc.3835

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Peer-reviewed

Ribosome Purification from an α-proteobacterium and rRNA Analysis by Northern Blot

α-变形杆菌核糖体的纯化及Northern印迹分析

LY Liang Yin

Caroline S. Harwood email

发布: 2020年12月05日第10卷第23期 DOI: 10.21769/BioProtoc.3835 浏览次数: 2477

评审: Alka MehraKenji SugiyamaAnonymous reviewer(s)

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Abstract

Ribosomes are an integral part of cellular life. They are complex molecular machines consisting of multiple ribosomal proteins and RNAs. To study different aspects of ribosome composition, many methods have been developed over the decades. Here, we describe how to purify ribosomes from the α-proteobacterium Rhodopseudomonas palustris. Following this protocol, RNA can be extracted from either purified ribosomes or directly from cell cultures, and ribosomal RNAs quantified using Northern blot. This protocol gives an example of studying ribosomes in a bacterium other than the commonly used E. coli. The challenge of performing Northern blots with rRNA is also addressed in detail.

Keywords: Ribosome profile (Ribosome profile)

capillary transfer (毛细管转移)

rRNA (核糖体RNA)

Northern blot (Northern印迹)

Rhodopseudomonas palustris (沼泽红假单胞菌)

Background

The fate of bacterial cells is closely linked to their ribosomes. Our recent study showed that active ribosomes play an important role in the survival mechanisms of nutrient-deprived R. palustris cells (Yin et al., 2019). The ribosomes are purified via a series of ultracentrifugations with a protocol optimized from the classical methods (Lawrence et al., 2016). Ribosomal RNA populations are detected with Northern blot employing a less frequently used capillary transfer system. The state of ribosomes can be greatly affected by the details of purification steps. These methods, described here in detail, should be of broad interest to researchers who study the translation apparatus in a wide variety of bacteria.

Materials and Reagents

Note: All reagents can be made de novo or purchased from different vendors as long as they are RNase free.

Centrifuge tubes, RNase-free (ThermoFisher AM12400 or equivalent). Use ultracentrifuge compatible tube for ultracentrifugation (please refer to the manual of reader’s ultracentrifuge for more information).
Screw-cap tube, 2 ml (Sigma-Aldrich, BR780758 or equivalent)
Costar Sterile Disposable Reagent Reservoirs (Fisher Scientific, catalog number: 07-200-128 )
HEPES-KOH, 1 M, pH 7.5 (Boston Bioproduct, catalog number: BBH-75-K )
MgCl₂, 1 M (Boston Bioproduct, catalog number: BM-670 )
NaCl, Molecular Biology Grade (Promega Corp, catalog number: H5271 )
Recombinant RNasin Ribonuclease Inhibitor (Promega Corp, catalog number: N2511 )
miRNeasy Mini Kit (Qiagen, catalog number: 217004 )
Agarose, molecular biology grade
UltraPure DNase/RNase-Free Distilled Water (Invitrogen, catalog number: 10977-023 )
Amersham Hybond-XL membrane (GE Life Sciences)
10x Tris-borate-EDTA (TBE) buffer (Thermo Fisher Scientific, catalog number: AM9863 )
20x SSC buffer (Sigma-Aldrich, catalog number: S6639 )
DIG RNA Labeling Mix (Sigma-Aldrich, catalog number: 11277073910 )
DIG Easy Hyb (Sigma-Aldrich, catalog number: 11603558001 )
DIG Wash and Block Buffer Set (Sigma-Aldrich, catalog number: 11585762001 )
Anti-Digoxigenin-AP, Fab fragments (Sigma-Aldrich, catalog number: 11093274910 )
CDP-Star (Sigma-Aldrich, catalog number: 11685627001 )
RNA Gel Loading Dye (2x) (ThermoFisher, catalog number: R0641 )
Buffer A1 (10x) (see Recipes)
Lysis buffer (see Recipes)

Equipment

Note: The working bench as well as electrophoresis, transfer and Northern blot systems should be cleaned with RNaseZap or equivalent to decontaminate RNase before use. Items below that do not have a catalog number listed can be obtained from any reliable supplier.

Mini-Beadbeater-24 (Bio Spec Products Inc., catalog number: 112011 )
Zirconia/Silica Beads, 0.1 mm dia (Bio Spec Products Inc., catalog number: 11079101z )
Beckman TL-1000 ultracentrifuge (discontinued model, can be substituted with equivalent ultracentrifuge with temperature-control). Please make sure to use compatible centrifuge tubes for ultracentrifugation.
Temperature-controlled tabletop centrifuge
Electrophoresis system suitable for agarose gel
A spectrometer capable of measuring UV absorbance, as well as matching quartz cuvettes
A short wavelength UV light
Pipette as well as matching pipetting tips. Any kind of pipette (manual, automatic, serological, etc.) works as long as they meet the requirement and are fit for use
Liquid nitrogen tank (and liquid nitrogen supply) or -80 °C freezer for storing bacterial strains
Incubator for bacterial growth

Procedure

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Yin, L. and Harwood, C. S. (2020). Ribosome Purification from an α-proteobacterium and rRNA Analysis by Northern Blot. Bio-protocol 10(23): e3835. DOI: 10.21769/BioProtoc.3835.