Anti-RFP tag Rabbit mAb conjugated Agarose Beads

Overview

Description

Anti-RFP tag Rabbit mAb conjugated Agarose Beads

Reactivity

Species independent

Tested applications

IP and Co-IP

Properties

Immunogen

Recombinant full length protein corresponding to RFP.

Isotype

Rabbit monoclonal, clone number: 3H5

Form

provided as 1ml of a 50% slurry suspended in PBS containing preservative (0.09% sodium azide).

*Azide may react with copper or lead in plumbing system to form explosive metal azides. Therefore, always flush plenty of water when disposing materials containing azide into drain.

Storage instruction

4°C

Host

Rabbit

Applications

Protocols

Protocol for Immunoprecipitation of RFP-Fusion Proteins from mammalian cell extract

 

1. Harvest cells

1). For one immunoprecipitation reaction the use of ~106 - 107 mammalian cells (approx. one 10-cm dish) expressing a RFP-tagged protein of interest is recommended. To harvest adherent cells, aspirate growth medium, add 1 ml ice-cold PBS to cells and scrape cells from dish. Transfer cells to a pre-cooled tube, spin at 500 g for 3 min at +4°C and discard supernatant. Wash cell pellet twice with ice-cold PBS, gently resuspending the cells. After washing:

 

 

2. Lyse cells

1). Resuspend cell pellet in 200 μl ice-cold lysis buffer by pipetting or using a syringe.

 

note: Supplement lysis buffer with protease inhibitors and 1 mM PMSF (not included).

optional for nuclear/chromatin proteins: Use RIPA buffer supplemented with 1 mg/ml DNase, 2.5 mM MgCl2, protease inhibitors and 1 mM PMSF (not included).

 

2). Place the tube on ice for 30 min with extensively pipetting every 10 min.

 

3). Centrifuge cell lysate at 20000x g for 10 min at +4°C. Transfer lysate to a pre-cooled tube. Add 300 μl dilution buffer to lysate. Discard pellet.

 

note: At this point cell lysate may be put at -80°C for long-term storage.

 

optional: Add 1 mM PMSF and protease inhibitors (not included) to dilution buffer.

 

3. Equilibrate beads

1). Vortex anti-RFP agarose beads and pipette 25 μl bead slurry into 500 μl ice-cold dilution buffer. Centrifuge at 2500x g for 2 min at +4°C. Discard supernatant and repeat wash twice.

 

4. Bind proteins

1). Add diluted lysate (step 2.3) to equilibrated anti-RFP agarose beads (step 3.1). If required, save 50 μl of diluted lysate for immunoblot analysis. Tumble end-over-end for 1 hour at 4°C.

 

2). Centrifuge at 2500x g for 2 min at +4°C. If required, save 50 μl supernatant for immunoblot analysis. Discard remaining supernatant.

 

5. Wash beads

1). Resuspend anti-RFP agarose beads in 500 μl ice-cold dilution buffer. Centrifuge at 2500x g for 2 min at +4°C. Discard supernatant and repeat wash twice.

optional: Increase salt concentration in the second washing step up to 500 mM.

 

6. Elute proteins

1). Resuspend anti-RFP agarose beads in 100 μl 2x SDS-sample buffer.

 

2). Boil resuspended anti-RFP agarose beads for 10 min at 95°C to dissociate immunocomplexes from anti-RFP agarose beads. anti-mCherry agarose beads can be collected by centrifugation at 2500x g for 2 min at 4°C and SDS-PAGE is performed with the supernatant.

 

3). optional instead of steps 6.1 and 6.2: elute bound proteins by adding 50 μl 0.2 M glycine pH 2.5 (incubation time: 30 sec under constant mixing) followed by centrifugation. Transfer the supernatant to a new tube and add 5 μl 1M Tris base pH 10.4 for neutralization. To increase elution efficiency this step can be repeated.