INSTRUCTIONS
Number
Description
20164
Pierce Magnetic RNA-Protein Pull-Down Kit, contains sufficient reagents for 20 desthiobiotinylation reactions with 50pmol of RNA and 20 pull-down reactions using 50pmol of labeled RNA and 50µL of magnetic beads Kit Contents:
Pierce RNA 3´ End Desthiobiotinylation Kit (20163); store at -20°C RBP Enrichment Module (20164Y); store at 4°C
Pierce Nucleic-Acid Compatible Streptavidin Magnetic Beads, 1mL, supplied at 10mg/mL in ultrapure water with 0.05% sodium azide
RNA Capture Buffer (1X), 10mL, 20mM Tris (pH 7.5), 1M NaCl, 1mM EDTA 20mM Tris, 5mL, pH 7.5ronan
undresdProtein-RNA Binding Buffer (10X), 1mL, 0.2M Tris (pH 7.5), 0.5M NaCl, 20mM MgCl 2, 1% Tween™-20 Detergent
Wash Buffer (1X), 10mL, 20mM Tris (pH 7.5), 10mM NaCl, 0.1% Tween-20 Detergent Biotin Elution Buffer, 1.5mL Glycerol, 50%, 0.5mL
HuR Monoclonal Antibody (Mou), 50µL, sufficient for detection of five Western blots
correct怎么读RNA Controls (20164Z); store at -20°C
Positive RNA Control (AR RNA), 250pmol (in 25µL), sufficient for five labeling and pull-down reactions
5´-CUGGGCUUUUUUUUUCUCUUUCUCUCCUUUCUUUUUCUUCUUCCCUCCCUA-3´ Negative RNA Control [poly(A)25 RNA], 250pmol (in 25µL), sufficient for five labeling and pull-down reactions
Storage: Upon receipt store Product No. 20163 and 20164Z at -20°C; store Product No. 20164Y at 4°C. Product No. 20163 is shipped on dry ice. Product No. 20164Y and 20164Z are shipped with an ice pack.
Table of Contents
Introduction ................................................................................................................................................................................. 2 Procedure Summary ..................................................................................................................................................................... 3 Important Product Information .................................................................................................................................................... 3 Additional Materials Required ..................................................................................................................................................... 4 Procedure for Enrichment of RNA-Binding Proteins Using RNA .............................................................................................. 4 Troubleshooting ........................................................................................................................................................................... 7 Related Thermo Scientific Products ....................................................................................................
........................................ 8 General References ...................................................................................................................................................................... 8 Product References . (8)
Pierce™ Magnetic RNA-Protein Pull-Down Kit
Introduction
The Thermo Scientific Pierce Magnetic RNA-Protein Pull-Down Kit provides reagents to efficiently enrich RNA-binding proteins (RBPs) using RNA end-labeled with desthiobiotin and Thermo Scientific Pierce Nucleic-Acid Compatible Streptavidin Magnetic Beads. Desthiobiotinylated target RNA directly enriches RBPs (or complexes) and provides an alternative to antibody capture of protein-RNA complexes. The kit offers veral advantages, including validated controls for labeling and the pull-down assay and compatibility with downstream applications such as Western blotting and mass spectrometry (MS).
The Thermo Scientific Pierce RNA 3´ End Desthiobiotinylation Kit us T4 RNA liga to attach a single desthiobiotinylated cytidine bisphosphate to the 3´ end of the RNA strand. End-labeling at the 3´ end does not interfere with RNA structure and is, therefore, more desirable than random incorpora
tion of labeled ribonucleotides. Each labeling reaction was designed for 50pmol of RNA; however, the labeling reactions may be scaled (1pmol to 1nmol have been tested), if necessary. The labeling reaction requires a 20-fold excess of desthiobiotinylated nucleotide with reaction incubation times from 30 minutes at 37°C for less complex RNA to overnight at 4-16°C for longer or more complex RNA. Optimization of the labeling efficiency for complex RNA structures is achieved by altering the RNA to nucleotide ratio, increasing the incubation time or by adding DMSO to the labeling reaction to relax the RNA structure.
The control system for the pull-down assay us 3´ untranslated-region androgen receptor (AR) RNA, poly(A)25 RNA and mammalian cell lysate. The proximal 3´ untranslated region (UTR) of AR RNA contains UC-rich regions for HuR and
Poly(C) Binding Proteins (CP1 and CP2). The RNA-binding proteins regulate mRNA stability (HuR) and mRNA turnover and translation (CP1 and CP2). The negative control RNA [poly(A)25 RNA] does not contain HuR or poly(C) BP binding sites. Incubation of A431 lysate with labeled AR UTR RNA enriches HuR RBP; incubation with only poly(A)25 RNA or beads does not enrich HuR RBP (Figure 1). However, the control system is versatile, and other cell lysates will work as sources of HuR.
Figure 1.Androgen receptor 3´-UTR RNA specifically pulls down HuR.
AR 3´-UTR RNA and poly(A)25 RNA (50pmol) were labeled using desthiobiotinylated
cytidine bisphosphate and T4 RNA liga using the kit procedure. Labeled RNA was
captured using 50µL of streptavidin magnetic beads in RNA Capture Buffer for
30 minutes at room temperature. Beads were washed twice in 20mM Tris (pH 7.5),
once in Protein-RNA Binding Buffer and 40µg of A431 extract was added. Samples
mandrakewere incubated for 45 minutes at 4°C, washed three times with Wash Buffer and eluted
after 15 minutes of incubation at 37°C with Biotin Elution Buffer. RNA pull-down
specificity was assd by Western blotting with samples normalized by volume and
bands detected using Thermo Scientific SuperSignal West Pico Substrate (Product No.
34080) and a 2-minute film exposure (L = lysate load; FT = flow-through; E = eluate).
Procedure Summary
The procedure for enrichment of RBPs has been optimized for ea of u (Figure 2). The RNA is first bound to the beads to orient the RNA for protein binding. RNA-bound beads are then equilibrated in Protein-RNA Binding Buffer before protein lysate is added. Beads are washed by adding the appropriate buffer, vortexing and parating on a magnetic stand. Additional salt, reducing agent or detergent may be added to the buffers to alter stringency. Samples may be eluted using non-denaturing
Biotin Elution Buffer or SDS-PAGE Loading Buffer.
Figure 2. Procedure summary schematic for the Thermo Scientific Pierce Magnetic RNA-Protein Pull-Down Kit.
Important Product Information
• Complete instructions for RNA labeling are included in the instruction booklet for the Pierce RNA 3´ End Desthiobiotinylation Kit (Product No. 20163).portlet
• Maintain a nuclea-free environment during the procedures and when working with the RNA intended for labeling. Wear gloves and only u reagents and plastics compatible with nucleic-acid manipulations.
•
The Pierce Nucleic Acid-Compatible Streptavidin Magnetic Beads are compatible with mass spectrometry becau of their low nonspecific binding. Do not freeze or dry the streptavidin magnetic beads. Freezing or drying will cau the beads to aggregate and lo binding activity.
• To minimize protein degradation, include protea inhibitors (e.g., Thermo Scientific Halt Protea Inhibitors or Pierce Protea Inhibitor Mini Tablets) in the cell lysate preparation.
• Boiling the magnetic beads in SDS-PAGE Reducing Sample Buffer is acceptable for single-u applications. Boiling will cau bead aggregation and loss of binding activity.
Additional Materials Required
•Target RNA for labeling
•Heated mixer or chiller
•Chloroform:isoamyl alcohol (24:1)
•Ethanol, absolute
•Cell Lysis Buffer (for preparation of cell lysate)
•Alternate Elution Buffer: SDS PAGE Reducing Sample Buffer (optional)
•Tris-buffered saline containing 0.05% Tween™-20 Detergent (TBS-T)
•Magnetic paration stand
•Western blotting reagents
•Nitrocellulo (Product No. 88014) or PVDF (Product No. 88585)
•Goat Anti-Mou IgG (H+L), HRP conjugated (Product No. 31430)
•SuperSignal™ West Pico Chemiluminescent Substrate (Product No. 34080)
•Electrophoresis apparatus
Procedure for Enrichment of RNA-Binding Proteins Using RNA
Note: Maintain nuclea-free conditions. Clean the work area, wear gloves and only u reagents and plastics compatible with nucleic acids.quintus什么意思
A.Pre-Washing Streptavidin Magnetic Beads (Optional)
Note: Nucleic acid-compatible beads were validated without pre-treatment of the beads.
1.Resuspend the beads in the original vial by gentle swirling or rotation.
2.Remove the amount to be treated and transfer to a nuclea-free tube.
3.Place tube on a magnetic stand to collect the beads against the sides of the tube.
4.Wash the beads twice with a 2X volume of 0.1M NaOH, 50mM NaCl (nuclea-free).
5.Wash the beads once in 100mM NaCl.
6.Continue with equilibration of magnetic beads for RNA capture (Section D).
B.Preparation of Cell Lysate
•Cell lysates may be prepared using standard lysis buffers (e.g., Thermo Scientific Pierce IP Lysis Buffer, M-PER Mammalian Protein Extraction Reagent and T-PER Tissue Protein Extraction Reagent).
•Protein(s) translated in vitro using human in vitro expression kits (e.g., Thermo Scientific 1-Step Human IVT Kits, Product No. 88881-9) may also be ud to test a protein’s ability to bind to an RNA target.
•Ensure the cell lysate protein concentration is greater than 2mg/mL, such that there is significant dilution into the Binding Reaction Buffer. If high salt or detergent interferes with the binding reaction, lysates may be buffer exchanged using Thermo Scientific Zeba Desalting Columns.
C.Label Target RNA
•Label the target RNA using the included Thermo Scientific Pierce RNA 3´ Desthiobiotinylation Kit, Product No. 20163.
Basic reaction components and amounts are provided in Table 1.
Table 1. Basic reaction components and protocol notes for the Thermo
Scientific Pierce RNA 3´ End Desthiobiotinylation Kit.
Component Volume (µL) Final Concentration
Nuclea-free Water 3 -
10X RNA Liga Reaction Buffer 3 1X
RNa Inhibitor 1 40U
Control RNA or Test RNA 5 50pmol
aboutBiotinylated Cytidine Bisphosphate 1 1nmol
T4 RNA Liga 2 40U
PEG 30% 15 15 %
Total Volume 30 -
Note: Add the PEG 30% to the reaction last and mix well. Incubate at the desired
temperature for the appropriate time. For the pull-down reaction, extract labeled
RNA with an equal volume of chloroform:isoamyl alcohol, followed by ethanol
precipitation.
D.Binding of Labeled RNA to Streptavidin Magnetic Beads
Note: U a range of 25-100pmol of RNA per 20-50µL of magnetic beads. The instructions below u a scale of 50pmol of RNA to 50µL of beads.
interaction
Note: Positive and negative RNA controls have been included in the kit. To determine specificity of ur-supplied RNA, u
a positive, negative and lysate-only control.
1.Add 50µL of streptavidin magnetic beads to a 1.5mL microcentrifuge tube.
2.Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant.
3.Wash with an equal volume of 20mM Tris (pH 7.5). Resuspend beads by pipetting or vortexing.
4.Repeat Steps 2 and 3.
5.Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant.
6.Add an equal volume of 1X RNA Capture Buffer. Resuspend beads by pipetting or vortexing.
7.Add 50pmol of labeled RNA to the beads. Mix gently by pipetting.
8.Incubate for 15-30 minutes at room temperature with agitation.
E.Binding of RNA-Binding Proteins to RNA
Note: The Protein-RNA Binding Buffer was developed as a starting point for binding reactions. Additi
onal reagents may also be added to the supplied binding buffer to enhance binding affinity and specificity. Ur-developed binding buffers are also compatible with this kit.
1.Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant.
2.Wash with an equal volume of 20mM Tris (pH 7.5). Resuspend beads by pipetting or vortexing.
3.Repeat Steps 1 and 2.
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4.Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant.
5.Dilute 10X Protein-RNA Binding Buffer to 1X (i.e., 10µL into 90µL of ultrapure water for each reaction).
6.Add 100µL of 1X Protein-RNA Binding Buffer to the beads and mix well.合肥健身
