RNA-Protein pull down

INSTRUCTIONS

RNA-Protein pull down

RNA-Protein pull down

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 7.5

Protein-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 (Mouse), 50μL, sufficient for detection of five Western blots

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 several 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 uses T4 RNA ligase 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 incorporation 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 uses 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). These 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.

RNA-Protein pull down

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 ligase 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

were 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 assessed 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 ease of use (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 separating 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.

RNA-Protein pull down

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).

? Maintain a nuclease-free environment during the procedures and when working with the RNA intended for labeling. Wear gloves and only use reagents and plastics compatible with nucleic-acid manipulations.

?

The Pierce Nucleic Acid-Compatible Streptavidin Magnetic Beads are compatible with mass spectrometry because of their low nonspecific binding. Do not freeze or dry the streptavidin magnetic beads. Freezing or drying will cause the beads to aggregate and lose binding activity.

? To minimize protein degradation, include protease inhibitors (e.g., Thermo Scientific Halt Protease Inhibitors or Pierce Protease Inhibitor Mini Tablets) in the cell lysate preparation.

? Boiling the magnetic beads in SDS-PAGE Reducing Sample Buffer is acceptable for single-use applications. Boiling will cause 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 separation stand

?Western blotting reagents

?Nitrocellulose (Product No. 88014) or PVDF (Product No. 88585)

?Goat Anti-Mouse 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 nuclease-free conditions. Clean the work area, wear gloves and only use reagents and plastics compatible with nucleic acids.

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 nuclease-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 (nuclease-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 used 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.

http://m.wendangku.net/doc/7a4cc620a8956bec0875e31d.htmlbel 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

Nuclease-free Water 3 -

10X RNA Ligase Reaction Buffer 3 1X

RNase Inhibitor 1 40U

Control RNA or Test RNA 5 50pmol

Biotinylated Cytidine Bisphosphate 1 1nmol

T4 RNA Ligase 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: Use a range of 25-100pmol of RNA per 20-50μL of magnetic beads. The instructions below use a scale of 50pmol of RNA to 50μL of beads.

Note: Positive and negative RNA controls have been included in the kit. To determine specificity of user-supplied RNA, use

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. Additional reagents may also be added to the supplied binding buffer to enhance binding affinity and specificity. User-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.

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.

7.Prepare a Master Mix of RNA-Protein Binding Reaction (Table 2).

Table 2. Reaction components for the Master Mix of RNA-Protein Binding Reaction.

Reagent Volume (μL) per 100μL reaction for control Range

10X Protein-RNA Binding Buffer 10 5-20μL

50% glycerol 30 0-50μL

Additional salts, etc. (variable) x xμL

Lysate (protein conc. > 2mg/mL 1-30 20-200μg

Nuclease-free water to 100 to 100μL

8.Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant.

9.Add 100μL of Master Mix to the RNA-bound beads. Mix by pipetting or gentle vortexing.

10.Incubate 30-60 minutes at 4°C with agitation or rotation.

F.Washing and Elution of RNA-Binding Protein Complexes

1.Place the tube into a magnetic stand to collect the beads against the side of the tube. Transfer the supernatant to a tube for

later analysis.

2.Wash with equal volume of 1X wash buffer (100μL).

3.Repeat Steps 1 and 2 two additional times. Save wash supernatants for analysis, if desired.

4.Place the tube into a magnetic stand to collect the beads against the side of the tube. Transfer the supernatant to a tube for

later analysis.

5.Add 50μL of Elution Buffer to the beads and mix well by vortexing. Incubate 15-30 minutes at 37°C with agitation.

6.Place the tube into a magnetic stand to collect the beads against the side of the tube.

7.Remove supernatant for downstream analysis.

8.If the downstream application is Western blotting, add reducing sample buffer to samples to 1X.

9.Heat the eluted samples for 5-10 minutes at 95-100°C. Electrophorese samples on a gel or store at -20°C until use.

10.For the control reaction, apply 30μL per lane.

G.Western Blot Analysis

Note: The control reactions have been optimized for use with SuperSignal West Pico Chemiluminescent Substrate. Include a cell lysate lane as a control to verify that the Western blot is properly functioning.

1.Separate the proteins by SDS-PAGE and transfer to nitrocellulose membrane.

2.Block the membrane in TBS-T containing 5% bovine serum albumin (BSA) at room temperature for 1 hour.

3.Prepare a solution containing 10μL of anti-HuR antibody in 10mL of TBS-T containing 5% BSA.

4.Incubate the membrane in the anti-HuR antibody for 4 hours at room temperature or overnight at 4°C.

5.Wash the membrane 5 times for 5 minutes each with TBS-T.

6.Dilute the Goat Anti-Mouse IgG (H+L) (HRP-conjugated, 1:20,000) into TBS-T containing 5% BSA.

7.Incubate the membrane in the diluted Goat Anti-Mouse IgG at room temperature for 1 hour.

8.Wash the membrane 5 times for 5 minutes each with TBS-T.

9.Incubate the membrane with chemiluminescent substrate at room temperature (e.g., SuperSignal West Pico

Chemiluminescent Substrate).

10.Immediately expose the membrane to X-ray film or a CCD camera for the appropriate exposures.

Note: The HuR band is located at ~36kDa.

Troubleshooting

Problem Possible Cause Solution

Degraded RNA Nuclease-free environment was

compromised

Clean work area and ensure all plastics are nuclease-free and from

unopened packages

mRNA from in vitro transcription

reaction was degraded

After transcription, ensure RNA is intact by gel electrophoresis

Inefficient recovery of RNA-binding protein Binding reaction was not

optimized

Optimize incubation time, temperature, salt and detergent for

binding reactions

Titrate amount of labeled RNA to protein lysate

Use a more concentrated lysate

Insufficient amount of magnetic

beads used for capture

Increase amount of magnetic beads for capture

Insufficient amount of RNA used

for capture

Increase amount of labeled RNA in reaction

Confirm good ligation efficiency

No recovery of RNA-binding protein Insufficient amount of target

protein in the sample

Increase amount of sample

Sample was not compatible with

binding reaction

Buffer exchange sample using Zeba? Desalting Columns

Binding reaction was not

optimized

Optimize incubation time, temperature, salt and detergent for

binding reactions

Titrate amount of labeled RNA to protein lysate

Use a more concentrated lysate

RNA binding protein had low

affinity for labeled RNA

Add crosslinking reagent (e.g., UV, etc.) after protein has bound

RNA

High nonspecific binding of RNA-binding protein Binding reaction was not

optimized

Optimize incubation time, temperature, salt, and detergent for

binding reactions

Insufficient washing stringency Increase stringency of wash buffer; add salt and/or detergent

Ratio of labeled RNA to lysate

was not optimized

Titrate labeled RNA with protein lysate

Reduce the concentration of lysate to ~2mg/mL

Low signal on Western blot Insufficient signal Increase amount of secondary antibody

Use a more sensitive chemiluminescent detection (e.g., SuperSignal

Dura or SuperSignal Femto Chemiluminescent Substrate)

Poor antibody quality Pre-screen antibody with cell lysate

Include cell lysate as a control on Western blot

Protein was insufficient in lysate Increase amount of sample

Identify alternate source of protein

Use a more concentrated lysate

Related Thermo Scientific Products

20163 Pierce RNA 3′ End Desthiobiotinylation Kit

20160 Pierce RNA 3′ End Biotinylation Kit

88881-9 1-Step Human In Vitro Translation Kits

88859-69 pT7CFE1 Cell-Free Expression Vectors

General References

Yeap, Bu. B., et al. (2002). Novel binding of HuR and Poly(C)-Binding Protein to a conserved UC-rich motif within the 3′ untranslated region of the androgen receptor messenger RNA. J Biol Chem277:27183-92.

Khanam, T., et al. (2006). Poly(A)-binding protein binds to A-rich sequences via RNA-binding domains 1+2 and 3+4. RNA Biology3:170-7.

Product References

Brown, R., et al. (1989). Protein measurement using bicinchoninic acid: Elimination of interfering substances. Anal Biochem180(1):136-9.

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