Anti-RFP tag Rabbit mAb conjugated Magnetic Beads

Overview

Description

anti-RFP Rabbit monoclonal antibdoy conjugated magnetic beads

Reactivity

All Species Expected

Tested applications

IP/Co-IP: 20 μL of beads slurry/400 μL of cell extract

Specificity

The antibody binds RFP at the N-terminal, Met-N-terminal, C-terminal and internal locations of fusion proteins.

Properties

Immunogen

Recombinant full length protein corresponding to RFP

Clonality

Rabbit monoclonal, clone number: 3H5

Isotype

Rabbit IgG

Form

Magnetic beads Conjugated anti-RFP antibody, in phosphate buffered saline (PBS), pH 7.3 (+/- 0.1), 0.02% sodium azide as preservative.

20%(v/v) slurry, total beads suspension volume of 1000 µL (200 µL packed beads)

Host

Rabbit

Applications

Application Image

Bead size:	~ 40 µm
Binding capacity:	10 µg of RFP fusion protein can be precipitated with 25 µL of beads suspension
Surface functional group density:	~2mg anti RFP antibody /mL

 

Protocols

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

This protocol is intended for immunoprecipitation of active proteins (native protein and fusion protein) for analysis by western immunoblot or activity assay.

 

Solutions and Reagents

10X Cell Lysis Buffer: 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin. Recommend adding 1 mM PMSF before use.

 

1X Wash Buffer: TBS (50 mM Tris HCl, 150 mM NaCl, pH 7.4)

3X SDS Sample Loading Buffer: 187.5 mM Tris-HCl (pH 6.8 at 25°C), 6% w/v SDS, 30% glycerol, 150 mM DTT, 0.03% w/v bromophenol blue

1. Preparing Cell Lysates

1. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS. Remove PBS and add 0.5 ml 1X ice-cold cell lysis buffer to each plate (10 cm) and incubate the plates on ice for 5 minutes.

2. Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.

3. Sonicate samples on ice three times for 5 seconds each.

4. Microcentrifuge for 10 minutes at 4°C, 14,000 x g, and transfer the supernatant to a new tube. If necessary, lysate can be stored at –80°C.

 

2. Immunoprecipitation

1. Take 1000 μL cell lysate and add 40 µl of the antibody conjugated magnetic beads suspension, incubate with rotation overnight at 4°C.

2. Wash the protein complex three times with a total of 20 packed beads volumes of TBS (50 mM Tris HCl, 150 mM NaCl, pH 7.4). (NOTE: if Co-IP interacting proteins are researched, please reduce the number of washes, and lower the ionic strength of the wash buffer.)

3. Elution for Downstream Analysis

Elution of the RFP fusion proteins - Two elution methods are recommended according to protein characteristics or further usage: 

 

Option A: Elution under acidic conditions with 0.1 M glycine HCl, pH 3.0. This is a fast and efficient elution method. Equilibration of the eluted proteins with neutralizing buffer (0.5 M Tris HCl, with 1.5 M NaCl, pH 7.4) may help preserve its activity. 

 

Option B: Elution with sample loading buffer under denaturing conditions for gel electrophoresis and immunoblotting.

 

A: Elution with 0.1 M Glycine HCl, pH 3.0 - The procedure should be performed at room temperature. Note：Do not leave the beads in this buffer more than 20 minutes.

 

1. Add 5 packed beads volumes of 0.1 M glycine HCl buffer, pH 3.0, to each sample and control beads complexes.

2. Incubate the samples and controls with gentle shaking or on a rotator for 5 minutes at room temperature. 

3. Place tube in the appropriate magnetic separator to collect the beads. Transfer the supernatants to fresh tubes containing 10 ml of 0.5 M Tris HCl, pH 7.4, with 1.5 M NaCl. Be careful not to transfer any beads. 

4. Repeat steps 1 - 3, pooling eluates in same tube. 

5. Equilibration of the eluted proteins with neutralizing buffer (0.5 M Tris HCl, with 1.5 M NaCl, pH 7.4) may help preserve its activity.

6. For immediate use, store the combined eluates at 2-8 °C. Store at –20 °C for long term storage. 

 

B: Elution with SDS-PAGE Sample Loading Buffer

1. Resuspend each sample with 20 µl 3X SDS sample buffer. Vortex.

2. Boil the sample and control tubes for 3 minutes at 95 – 100°C. 

3. Place tubes in the appropriate magnetic separator to collect the beads. Transfer the supernatants to fresh tubes. The samples and controls are ready for loading on SDS-PAGE and immunoblotting using anti-RFP or specific antibodies against the fusion protein.