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Protocol - RNA-protein interaction

RNA binding assay REMSA (RNA electrophoresis shift assay)

RNA-protein interaction
Category:  Molecular Biology
Creation date: Jun 16, 2014
Last revision: May 26, 2015
Author(s):  Pasquale Barba, Laura Pisapia and Giovanna Del Pozzo
Contact
Name: Giovanna Del Pozzo
Address: IGB, Via Pietro Castellino 111,Naples
Phone: 0816132309
Email: giovanna.delpozzo@igb.cnr.it

 

Figure legend:

REMSA experiments performed using 3-DRA (lane 1) and 3-DQA1 (lane 3) riboprobes; lanes 2 and 4 show the digestion of riboprobes with T1 RNase. Lanes 5 and 15 show bands of interaction of M14 S100 cytoplasmic extract with 3-DRA, lanes 10 and 18 with 3-DQA1; competition experiments of 3-DRA binding were performed using cold 3-DRA (lanes 6 and 7), cold 3-DQA1 (lanes 8 and 9) and poly(U) homopolymers (lanes 16 and 17). Competition experiments of 3-DQA1 binding were performed using cold 3-DRA (lanes 11 and 12), cold 3-DQA1 (13 and 14) and poly(U) homopolymers (lanes 19 and 20). (B) REMSAs experiments carried out using 3-DRB1 (lane 21) and 3-DQB1 (lane 22) riboprobes. Lanes 22 and 24 show the digestion of riboprobes with T1 RNase; lanes 27 and 32 show bands of interaction of M14 extract with 3-DRB1 and 3-DQB1. Competition experiments of 3-DRB1 binding were performed using cold 3-DRA (lane 25 and 26) and cold 3-DQA1 (28 and 29). Competition experiments of 3-DQB1 binding were performed using cold 3-DRA (lanes 30 and 31) and cold 3-DQA1(33 and 34) (Corso et al. 2011).

Steps

Description Temperature Time Note
Incubate 20 μg S100 cytoplasmic proteins with with 100000 cpm of 32P-UTP-labelled riboprobe in final volume of 20 μl of binding buffer
22°C
30 min
Treat each samples with heparin at a final concentration of 5 mg/ml
22°C
10 min
Add 1-5 U RNase T1 ( from Aspergillus Okazaky)
22°C
10 min
Add 10 μl of 6x Loading Buffer
22°C
2 min
Load samples on native 6% PAGE and run the gel at 200 VOLT using 0,3 X TBE buffer
22°C
3 hrs
Use MAXI PROTEAN apparatus
Dry gel
22°C
45 min
Acquire the radiactive image by Typhoon analysis (GE Healthcare).
22°C
over night

Other informations

Add two samples control:  1) the undigested riboprobe ; 2) the  riboprobe digested with RNase without extract

When required riboprobes were refolded by heating to 70°C for 5 min and slow-cooling
to room temperature over 1 h before use in binding buffer

Competition assays were performed by pre-incubation of cold riboprobe ( scalar quantity : es. 0.05 and 0.5 ug ) with protein extract for 15’ before to add labelled riboprobe

For antibody supershift assays:
0.5, 2 and 6 ug of polyclonal or monoclonal antibody
were pre-incubated with cell extracts prior to the
addition of labelled riboprobe .

6 ug of IgG were used in a  control sample

 

Quality validation: Yes

Validation info

The protocol has been used to produce data published in peer-reviewed journals.

 

Citations
Pisapia L, Cicatiello V, Barba P, Malanga D, Maffei A, Hamilton RS, Del Pozzo G. Co-regulated expression of alpha and beta mRNAs encoding HLA-DR surface heterodimers is mediated by the MHCII RNA operon. Nucleic Acids Res. 2013;41:3772-3786.
Corso C, Pisapia L, Citro A, Cicatiello V, Barba P, Cigliano L, Abrescia P, Maffei A, Manco G, Del Pozzo G. EBP1 and DRBP76/NF90 binding proteins are included in the major histocompatibility complex class II RNA operon. Nucleic Acids Res. 2011;39:7263-7275.

 

Related Protocols:

Solutions

  • 1x binding buffer

    Reagents F-concentration Quantity I-concentration
    HEPES pH 7.6
    10 mM
    MgCl2
    3 mM
    glycerol
    5%
    KCl
    40 mM
    DTT
    1 mM
    protease inhibitors
    1x

    Note

  • GEL

    Reagents F-concentration Quantity I-concentration
    acrilammide-bis (29:1)
    6%
    10 ml
    40%
    TBE (Tris Borate buffer )
    0,3X
    1,5 ml
    10X
    Ammonio Persolfato
    300 μl
    10%
    TEMED
    30 μl

  • Loading Buffer 6X

    Reagents F-concentration Quantity I-concentration
    Tris HCl pH 7,5
    30 mM
    saccarosio
    40%
    TBE (Tris Borate buffer )
    5X
    bromofenolo
    0,2%
    EDTA pH 8
    0,001M

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