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Protocol for Anti Ago-RNA Immunoprecipitation from mammalian cells using the RIP kit

Procedure:

Reagents required:

Catalogue Number
Description
RIP
Imprint® RNA Immunoprecipitation Kit
SAB4200085
Monoclonal Anti-AGO2 antibody produced in rat, clone 11A9
R9255
Anti-Rat IgG (whole molecule) antibody produced in rabbit
(aka, rabbit anti-rat)
I4131
IgG from rat serum
09691
Ammonium acetate solution, 5M
Ambion AM9520
Linear Acrylamide (5 mg/ml)
I9516
2-Propanol
   
For harsh/stringent
wash solution:

 
I8896
IGEPAL® CA-630
S5150
Sodium chloride solution 5M

Prep cells

1.    Grow cells to ~80% confluency.  You will need ~2 million cells/RIP, or ~4 million cells for IgG (neg control) & Ago2 RIP

2.    Rinse cells with HBSS (volume = culture medium)

3.    If cells attached, release in trypsin-EDTA ~5 min and quench with 4-5 volumes serum-containing medium

4.    Count cells, aliquot 2 million cells/ tube, and pellet at 4 oC, 1000rpm, 5 min.  Discard supernatant.

5.    Wash 2x with ice cold PBS (volume = medium removed).

6.    Discard supernatant & add 200µl mild or harsh lysis buffer + Protease Inhibitor Cocktail (PIC) (P8340) +    
       Ribonuclease Inhibitor (RNase inhibitor)(R1158)/cell pellet

 

Reagent:       per RIP 2.1 RIPs
Lysis buffer     0.2 0.42 ml
PIC (protease inhibitor cocktail) 2.0 4.2 µl
RNase inhibitor     0.8 1.68 µl

7.    Incubate on ice 15' (or store o/n at -80 oC)

8.    Pellet debris at 4 oC, 16,000g, 10 min

9.    Transfer sup to fresh tube

10.  Remove 10 µl from each cell lysate for 5% input and store on ice

Prep beads

1.    Add 40µl Protein A Magnetic Beads to 200µl wash buffer in 1.5ml tube for 2 RIPs (IgG & Ago2)

2.    Place tubes on magnetic stand & remove liquid

3.    Wash 1x 200µl wash buffer

4.    Resuspend in 200µl wash buffer

5.    Add 5 µg rabbit Anti-Rat IgG (whole molecule) antibody produced in rabbit (R9255)

6.    Incubate at RT with rotation for 30 min.

7.    Spin briefly & remove liquid on magnetic stand

8.    Wash 2x with 1ml wash buffer.

9.    Resuspend in 200µl wash buffer & split into 2 x 100µl

10.  Add 2.5 µg rat IgG (I4131) or Monoclonal Anti-AGO2 antibody produced in rat, clone 11A9 (SAB4200085).

11.  Incubate at RT with rotation for 30 min.

12.  Spin briefly & remove liquid on magnetic stand.

13.  Wash 2 times with 0.5ml wash buffer.

14.  Remove wash buffer completely on magnetic stand.

Doing RIP

1.    Prep IP Buffer:

Reagent:

 

 

 

Proportions 2.1 x mild 2.1 x harsh
Wash buffer

 

 

1 0.63 ml 1.68 ml
PIC (protease inhibitor cocktail) 10 6.3 µl 16.8 µl
RNase inhibitor

 

 

4 2.52 µl 6.72 µl

 

1.    Resuspend bead pellets in 0.2ml IP Buffer.

2.    Verify 5% input reserved from each cell lysate and stored on ice

3.    Add 0.1 ml IP Buffer/cells in mild lysis or 0.6 ml/cells in harsh lysis

4.    Transfer diluted lysate to resuspended beads

5.    Incubate at 4 oC with rotation O/N

Wash

1a. For mild washing, wash 5x by adding 1ml wash buffer, vortex, spin briefly, collect beads on magnet, & remove
     supernatent

1b. For harsh or stringent washing, wash 1x 1ml wash buffer, 2x 1ml stringent wash buffer, and 2x 1ml standard    
     wash buffer
Stringent wash buffer:

Reagent Initial Final 10 ml
Wash buffer   1 10 ml
100% Igepal 0.05 1 0.095 ml
5 M NaCl 0.15 0.9 1.5 ml



2. After last wash, resuspend in 0.2ml wash buffer

3. Bring reserved input samples to 0.2ml with wash buffer  

Purify RNA

1.    Add 0.5ml Tri Reagent (T9424) to each 0.2ml RIP or input

2.    Add 0.1ml chloroform (C2432) to each, vortex thoroughly, and spin at 4 oC, 16,000g, 10 min.

3.    Prepare 1.5ml tubes containing:

        6 µl linear acrylamide (Ambion; 5mg/ml)

        60 µl 5M ammonium acetate (09691)

        600 µl 2-propanol (I9516)

4.    Transfer aqueous to tubes from above, vortex, & precipitate at -80 oC at least 1 hour

5.    Thaw tubes from -80 oC on ice

6.    Spin at 4 oC, 16,000g, 10 min.

7.    Carefully pour off supernatant

8.    Wash once with 0.5ml 70% ethanol

9.    Spin at 4 oC, 16,000g, 10 min.

10.  Carefully pour off sup and dry pellets in a laminar flow hood

11.  Rehydrate each in 10µl RNase-free water

Reagents required:

Imprint RNA Immunoprecipitation Kit RIP
RIP Kit components required:  
Mild Lysis Buffer B0314
Harsh Lysis Buffer B0439
RIP Wash Buffer B0564
Protein A Magnetic Beads B0689
Ribonuclease Inhibitor R1158
Protease Inhibitor Cocktail P8340

 

Additional reagents required:

Monoclonal Anti-AGO2 antibody produced in rat, clone 11A9 SAB4200085
Anti-Rat IgG (whole molecule) antibody produced in rabbit R9255
IgG from rat serum I4131
TRI Reagent T9424
Chloroform C2432
Ammonium acetate solution, 5M 09691
2-Propanol I9516
Linear Acrylamide (5 mg/ml) Ambion AM9520

For making harsh/stringent wash solution:

IGEPAL CA-630 I8896
Sodium chloride solution, 5M S5150

Materials

     
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