RAB-IP Anti-rabbit IgG (HRP) for IP/CoIP

Overview

Description

RAB-IP™ Anti-rabbit IgG (native conformation antibody specific) for IP/CoIP (HRP Conjugated),

so,

RAB-IP™ Anti-rabbit IgG for IP/CoIP (HRP Conjugated) only recognize native (non-reduced) primary antibodies (from rabbit) and therefore the bands of heavy chain (50kD) and light chain (25kD) are eliminated.

Reactivity

Rabbit

Tested applications

WB : 1/1000-1/2000. Optimal dilutions should be determined by the end user.

Product Picture

Specificity

This secondary antibody is specific to native rabbit polyclonal/monoclonal antibody

Properties

Immunogen

The antibody was developed in goat using the nature rabbit IgG as the immunogen and then develop monoclonal antibody.

Clonality

Monoclonal, clone number: 3B8

Isotype

Goat IgG

Form

The antibody in 10 mM phosphate buffered saline (PBS), pH 7.4, 50% glycerol with 10 mg/mL (1% w/v) BSA (IgG free, protease free) as a stabilizer and 0.01% (w/v) thimerosal as preservative.

Storage instruction

Store at -20°C, Avoid freeze / thaw cycle. Do not aliquot the antibody.

Conjugation

Horseradish Peroxidase (HRP)

Host

Goat

Product Sheet

Applications

IP/CoIP Image

 Figure 1. RAB-IP™ Anti- rabbit IgG for IP (HRP) secondary antibody images: Western blotting.

 

IP conditions:

target protein TBP was immunoprecipitated from 0.5 mL cell lysate of 1x107 Hela cells with 5 µg anti-human TBP rabbit monoclonal antibody and protein A/G agarose beads (Engibody, IF0001).

 

WB conditions: 

the immunoprecipitated antigen-antibody complex should be boiled for 5-10 minutes in SDS sample buffer with a increase in SDS amount if required, make sure that the complex is fully reduced and denatured before downstream WB.

then electrophoresis, transferred to a PVDF membrane, and incubate with an anti- TBP rabbit monoclonal antibody.

 

Secondary Antibody Detection:

Lane 1: Detection with RAB-IP™ Anti-rabbit IgG for IP (HRP) (CAT: IF9203)

The heavy and light chains can not be seen, confirming that although the immunoprecipitating heavy and light-chains are present, detects only native antibody and not denatured heavy and light-chains.

 

Lane 2: Detection with a traditional goat anti-rabbit IgG (H&L) (HRP) secondary antibody (CAT: AT0097)

 

Work Flow Figure 1

 

Figure 2. A schematic representation of IP-WB workflow by using RAB-IP™ secondary antibody.

Using primary antibody coupled beads

RAB-IP™ Anti-rabbit IgG for IP (HRP Conjugated) only recognize native (non-reduced) primary antibodies (from rabbit) and therefore the bands of heavy chain and light chain are highly minimized, if the immunoprecipitated antigen-antibody complex is fully reduced and denatured before downstream WB.

Work Flow Figure 2

Figure 3. A schematic representation of IP-WB workflow by using RAB-IP™ secondary antibody.

Using protein A/G Beads + primary antibody

RAB-IP™ Anti-rabbit IgG for IP (HRP Conjugated) only recognize native (non-reduced) primary antibodies (from rabbit) and therefore the bands of heavy chain and light chain are highly minimized, if the immunoprecipitated antigen-antibody complex is fully reduced and denatured before downstream WB.